Publications by authors named "Gianna Rossi"

28 Publications

  • Page 1 of 1

Bisphenol A Deranges the Endocannabinoid System of Primary Sertoli Cells with an Impact on Inhibin B Production.

Int J Mol Sci 2020 Nov 26;21(23). Epub 2020 Nov 26.

Department of Applied Clinical and Biotechnological Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

Bisphenol A (BPA) is an endocrine disruptor that negatively affects spermatogenesis, a process where Sertoli cells play a central role. Thus, in the present study we sought to ascertain whether BPA could modulate the endocannabinoid (eCB) system in exposed mouse primary Sertoli cells. Under our experimental conditions, BPA turned out to be cytotoxic to Sertoli cells with an half-maximal inhibitory concentration (IC) of ~6.0 µM. Exposure to a non-cytotoxic dose of BPA (i.e., 0.5 μM for 48 h) increased the expression levels of specific components of the eCB system, namely: type-1 cannabinoid (CB) receptor and diacylglycerol lipase-α (DAGL-α), at mRNA level, type-2 cannabinoid (CB) receptor, transient receptor potential vanilloid 1 (TRPV1) receptors, and DAGL-β, at protein level. Interestingly, BPA also increased the production of inhibin B, but not that of transferrin, and blockade of either CB receptor or TRPV1 receptor further enhanced the BPA effect. Altogether, our study provides unprecedented evidence that BPA deranges the eCB system of Sertoli cells towards CB- and TRPV1-dependent signal transduction, both receptors being engaged in modulating BPA effects on inhibin B production. These findings add CB and TRPV1 receptors, and hence the eCB signaling, to the other molecular targets of BPA already known in mammalian cells.
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http://dx.doi.org/10.3390/ijms21238986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730056PMC
November 2020

Repeated hyperstimulation affects the ultrastructure of mouse fallopian tube epithelium.

J Reprod Dev 2020 Aug 28;66(4):387-397. Epub 2020 Apr 28.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

Controlled ovarian hyperstimulation (COH) is routinary used in assisted reproductive technologies (ARTs) to increase the yields of mature oocytes. The possibility that patients with a history of failures or poor-responders may develop side-effects following these treatments is still debated. Epidemiological studies reported controversial results about pregnancy outcome and the risk of developing gynecological cancers. By using a mouse model, here we compared the ultrastructural features of fallopian tubes (FTs) obtained from mice undergoing or not (control, CTR) four (4R) and eight (8R) rounds of gonadotropin stimulation. Although the morphological characteristics of oviductal layers seemed unaffected by repeated treatments, dose-response ultrastructural alterations in the ampulla appeared in the 4R group and even more in the 8R group. The targets were oviductal ciliated (CCs) and non-ciliated (NCCs) cells, which showed damaged mitochondria and glycogen accumulations in the cytoplasm. The drastic reduction of CCs, evident after 4R, was supported by the absence of cilia. After 8R, glycogen granules were significantly reduced and massive degeneration of mitochondria, which appeared swollen and/or vacuolated, occurred in NCCs. Moreover, disintegrated mitochondria were found at the periphery of mitophagic vacuoles with evident signs of cristolysis. The morphometric analysis evidenced a significant increase in the density and frequency of damaged mitochondria after 4R and 8R. The absence of cilia, necessary to sustain oviductal transport of oocytes, spermatozoa and embryos, may originate from either mitochondrial dysfunction or glycogen consumption. These results suggest that repeated COH treatments could induce alterations impairing fertilization and embryo transport toward the uterus.
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http://dx.doi.org/10.1262/jrd.2019-147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470905PMC
August 2020

The (endo)cannabinoid signaling in female reproduction: What are the latest advances?

Prog Lipid Res 2020 01 18;77:101019. Epub 2019 Dec 18.

Department of Medicine, Campus Bio-Medico University of Rome, 00128 Rome, Italy; European Center for Brain Research (CERC)/Santa Lucia Foundation, Via del Fosso di Fiorano, 64 - 00143 Rome, Italy. Electronic address:

Cannabis extracts like marijuana have the highest consumption rate worldwide. Yet, their societal acceptance as recreational and therapeutic drugs could represent a serious hazard to female human reproduction, because cannabis ingredients [termed (phyto)cannabinoids] can perturb an endogenous system of lipid signals known as endocannabinoids. Accumulated evidence on animal models and humans has demonstrated a crucial role of these endogenous signals on different aspects of female reproduction, where they act through an ensamble of proteins that synthesize, transport, degrade and traffic them. Several reports have recently evidenced the potential role of endocannabinoids as biomarkers of female infertility for disease treatment and prevention, as well as their possible epigenetic effects on pregnancy. The purpose of this review is to provide an update of data collected in the last decade on the effects of cannabinoids and endocannabinoids on female reproductive events, from development and maturation of follicles and oocytes, to fertilization, oviductal transport, implantation and labor. In this context, a particular attention has been devoted to the ovary and the production of fertilizable oocytes, because recent studies have addressed this hot topic with conflicting results among species.
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http://dx.doi.org/10.1016/j.plipres.2019.101019DOI Listing
January 2020

Increased levels of proapoptotic markers in normal ovarian cortex surrounding small endometriotic cysts.

Reprod Biol 2019 Sep 12;19(3):225-229. Epub 2019 Aug 12.

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy. Electronic address:

Endometriosis can impair fertility by reducing ovarian reserve and the production of good-quality oocytes. The surgical removal of endometriotic lesions is generally recommended for women who wish to conceive. In this paper we studied whether ovarian cortex adjacent to excised small (diameter ≤ 4 cm) endometriotic cyst (here referred as Cortex Surrounding Endometriotic Cyst, CSEC) showed signs of tissue damages by evaluating the expression of proteins involved in DNA repair and apoptosis. To this end, phosphorylated H2A.X, Chk1 and 2, ATM and ATR, Bcl-2, Bid, phosphorylated and total p53, caspases (9, 8 and 3), XIAP, phosphorylated and total NFκB were analyzed by western blot. Results showed that caspase 8, XIAP, p53/p-p53 and NFκB were more abundantly expressed in all samples of CSEC group in comparison with ovarian cortex of controls. Conversely, the levels of the other proteins were comparable between the two groups. In conclusion, these results suggest that NFκB, caspase 8 and p53/p-p53 elevated expressions in samples of CSEC can be considered as an early sign of tissue injury, indicating that ovarian cortex is already sensitized to apoptosis and inflammation. Therefore, excision of EC should occur very early, to avoid further ovarian damages.
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http://dx.doi.org/10.1016/j.repbio.2019.08.002DOI Listing
September 2019

Role of Major Endocannabinoid-Binding Receptors during Mouse Oocyte Maturation.

Int J Mol Sci 2019 Jun 12;20(12). Epub 2019 Jun 12.

European Center for Brain Research, Santa Lucia Foundation IRCCS, 00142 Rome, Italy.

Endocannabinoids are key-players of female fertility and potential biomarkers of reproductive dysfunctions. Here, we investigated localization and expression of cannabinoid receptor type-1 and -2 (CBR and CBR), G-protein coupled receptor 55 (GPR55), and transient receptor potential vanilloid type 1 channel (TRPV1) in mouse oocytes collected at different stages of in vivo meiotic maturation (germinal vesicle, GV; metaphase I, MI; metaphase II, MII) through qPCR, confocal imaging, and western blot. Despite the significant decrease in CBR, CBR, and GPR55 mRNAs occurring from GV to MII, CBR and GPR55 protein contents increased during the same period. At GV, only CBR was localized in oolemma, but it completely disappeared at MI. TRPV1 was always undetectable. When oocytes were in vitro matured with CBR and CBR but not GPR55 antagonists, a significant delay of GV breakdown occurred, sustained by elevated intraoocyte cAMP concentration. Although CBRs antagonists did not affect polar body I emission or chromosome alignment, GPR55 antagonist impaired in ~75% of oocytes the formation of normal-sized MI and MII spindles. These findings open a new avenue to interrogate oocyte pathophysiology and offer potentially new targets for the therapy of reproductive alterations.
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http://dx.doi.org/10.3390/ijms20122866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627642PMC
June 2019

Modulation of Endocannabinoid-Binding Receptors in Human Neuroblastoma Cells by Tunicamycin.

Molecules 2019 Apr 11;24(7). Epub 2019 Apr 11.

Department of Medicine, Campus Bio-Medico University of Rome, 00128 Rome, Italy.

Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on -linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of -linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as -acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB₁ and CB₂) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB₁ and CB₂, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with -glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB₁ (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB₁ with GlcNAc residues, and showed that tunicamycin reduced CB₁ membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support -linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors' expression and localization, in particular for CB₁.
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http://dx.doi.org/10.3390/molecules24071432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479803PMC
April 2019

Tebuconazole and Econazole Act Synergistically in Mediating Mitochondrial Stress, Energy Imbalance, and Sequential Activation of Autophagy and Apoptosis in Mouse Sertoli TM4 Cells: Possible Role of AMPK/ULK1 Axis.

Toxicol Sci 2019 05;169(1):209-223

Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, L'Aquila, Italy.

Tebuconazole and Econazole are triazole and imidazole fungicides currently used worldwide. Although their reproductive toxicity in mammals has been described, their effect on male reproductive systems has been poorly investigated. As humans may be exposed to different azole compounds simultaneously, the combinational in vitro toxicity of Tebuconazole and Econazole (MIX) in mouse Sertoli TM4 cells was investigated. This study demonstrates that Tebuconazole (40 µM) and Econazole (20 µM) act synergistically in mediating decrease of mitochondrial membrane potential (ΔΨm) and changes in mitochondrial morphology. These events were associated with ATP depletion, cell cycle arrest, and sequential activation of autophagy and apoptosis. Remarkable differences on other parameters such as AMP/ATP ratio and adenylate energy charge were observed. Pharmacological inhibition of autophagy by bafilomycin A1 leads to enhanced MIX-induced apoptosis suggesting an adaptive cytoprotective function for MIX-modulated autophagy. Finally, a possible role of AMPK/ULK1 axis in mediating adaptive signalling cascades in response to energy stress was hypothesized. Consistently, ULK1 Ser 555 phosphorylation occurred in response to AMPK (Thr 172) activation. In conclusion, Tebuconazole and Econazole combination, at concentrations relevant for dermal and clinical exposure, induces a severe mitochondrial stress in SCs. Consequently, a prolonged exposure may affect the ability of the cells to re-establish homeostasis and trigger apoptosis.
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http://dx.doi.org/10.1093/toxsci/kfz031DOI Listing
May 2019

VEGFR2 Expression Is Differently Modulated by Parity and Nulliparity in Mouse Ovary.

Biomed Res Int 2018 16;2018:6319414. Epub 2018 Sep 16.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

Parity and nulliparity exert opposite effects on women's health, as parity is considered a protective factor for several reproductive diseases. This study is aimed at determining if ovarian VEGF and VEGFR2 expression are differently modulated in the ovaries of parous and nulliparous mice. To this end primiparous and nulliparous fertile mice were sacrificed at postovulatory stage. Whole ovaries, corpus luteum, and residual stromal tissues were analyzed to assess VEGF/VEGFR2 expression levels. Ovarian mRNA amounts of ( and ) and were comparable between primiparous and nulliparous mice; both isoforms and receptor were accumulated mainly in corpus luteum tissues. VEGF 120 and 164 protein accumulation and distribution mirrored that of mRNA. Conversely, VEGFR2 protein content was significantly higher in ovaries of nulliparous mice and was more efficiently phosphorylated in ovaries of primiparous mice. In both groups, VEGFR2 was preferentially expressed in corpus luteum, while its phosphorylated form was equally distributed in two somatic compartments. We suggest that parity influences VEGFR2/phospho-VEGFR2 expression and tissue distribution. This difference could be part of a more complex mechanism that at least in mice is activated after the first pregnancy and likely aims to preserve female health.
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http://dx.doi.org/10.1155/2018/6319414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166384PMC
January 2019

Increased rounds of gonadotropin stimulation have side effects on mouse fallopian tubes and oocytes.

Reproduction 2018 03 4;155(3):245-250. Epub 2018 Jan 4.

Department of LifeHealth and Environmental Sciences, University of L'Aquila, L'Aquila, Italy

In this study, it was evaluated if increased rounds of gonadotropin stimulation could affect in mice: (i) expression levels of proteins regulating cell cycle and DNA repair in fallopian tubes and (ii) meiotic spindle morphology of ovulated oocytes. To this end, adult female mice were subjected or not (Control) to 6 or 8 rounds of gonadotropin stimulation. Ovulated oocytes were incubated with anti A/B tubulin to evaluate spindle morphology. Fallopian tubes were analyzed to detect Cyclin D1, phospho-p53/p53, phospho-AKT/AKT, phospho-GSK3B/GSK3B, SOX2, OCT3/4, phospho-B-catenin/B-catenin, phospho-CHK1 and phospho-H2A.X protein levels. After 6 rounds, Cyclin D1, p53 and phospho-p53 contents were higher than Control. After 8 rounds, the contents of phosphorylated AKT, GSK3B and p53 as well as of total p53, Cyclin D1 and OCT3/4 significantly increased in comparison with Control. Conversely, SOX2 and B-catenin were similarly expressed among all experimental groups. The finding that phospho-CHK1 and phospho-H2A.X protein levels were undetectable supported the absence of extensive DNA damage. Oocytes number and percentage of normal meiotic spindles drastically decreased from 6 rounds onward. Altogether, our results demonstrated that 6 and 8 cycles of gonadotropin stimulation reduce mouse reproductive performances by inducing over-expression and over-activation of proteins controlling cell cycle progression in fallopian tubes and by impairing oocyte spindle.
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http://dx.doi.org/10.1530/REP-17-0687DOI Listing
March 2018

Mancozeb affects mitochondrial activity, redox status and ATP production in mouse granulosa cells.

Toxicol In Vitro 2015 Dec 25;30(1 Pt B):438-45. Epub 2015 Sep 25.

Dept. of Life, Health and Environmental Sciences, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy.

Background: Mancozeb (MZ) is a fungicide that belongs to the subclass of metal (Mn/Zn) ethylene-bis-dithiocarbamate pesticides. In mouse and human granulosa cells (GCs) exposed to MZ (0.01 μg/ml), morphological modifications and significant alterations of p53 expression level in comparison with control GCs were recorded.

Objectives: To investigate if MZ (0.01 μg/ml) induces oxidative stress and alters energy metabolism in exposed mouse GCs.

Results: Following fungicide exposure, GCs showed low p53 content, a depolarized mitochondrial membrane potential (ΔΨm), as well as low ATP and reduced glutathione (GSH) levels associated with increased reactive oxygen species (ROS) generation. No remarkable differences on other parameters such as ATP/ADP ratio, energy charge, as well as induction of apoptosis and DNA damage were found. The activation of AKT and PDK1 kinases in MZ-treated cells was observed. Inhibition of ROS generation by the antioxidant N-acetylcysteine (NAC) restored a normal expression level of p53.

Conclusions: Our results demonstrate that the low dose of MZ here used induces a mild oxidative stress in GCs, and provides evidence for the possible involvement of AKT/PKB signaling pathway in triggering adaptive and survival response.
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http://dx.doi.org/10.1016/j.tiv.2015.09.018DOI Listing
December 2015

Endocannabinoid signaling in mammalian ovary.

Eur J Obstet Gynecol Reprod Biol 2014 Jul 18;178:6-11. Epub 2014 Apr 18.

Center of Integrated Research, Campus Bio-Medico University of Rome, Rome, Italy; European Center for Brain Research (CERC)/Santa Lucia Foundation, Rome, Italy. Electronic address:

The role of the endocannabinoid system (ECS) in mammalian reproduction is a rather active field of research, due to its potential exploitation to combat human infertility. Available data shows that the aberrant endocannabinoid signaling negatively affects embryo development, implantation and pregnancy. Although many efforts have been devoted to a better understanding of the ECS in these steps of female reproduction, very little is known about its role in regulating ovarian follicle development and production of mature oocytes. This is the subject of the present review where we discuss current knowledge about the impact and potential exploitation of the ECS and endocannabinoid signaling in mammalian ovary and folliculogenesis.
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http://dx.doi.org/10.1016/j.ejogrb.2014.04.011DOI Listing
July 2014

Repeated ovarian stimulation does not affect the expression level of proteins involved in cell cycle control in mouse ovaries and fallopian tubes.

J Assist Reprod Genet 2014 Jun 12;31(6):717-24. Epub 2014 Mar 12.

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

Purpose: To understand if repeated cycles (2-4 rounds) of gonadotropin stimulation could affect intracellular localization/content of proteins controlling cell cycle progression in mouse fallopian tubes (FT) and ovaries.

Methods: FT and ovaries of estrous mice (control) and of stimulated mice were analyzed to detect Oct-3/4, Sox-2, p53, β-catenin, pAKT and cyclin D1 localization/content. Spindles and chromosome alignment were analyzed in ovulated oocytes.

Results: After round 4, FT and ovaries of control and stimulated groups showed no differences in Oct-3/4, Sox-2 and β-catenin localization nor in Oct-3/4, Sox-2, p53, β-catenin and pAKT contents. Cyclin D1 level increased significantly in FT of treated mice. Oocytes number decreased meanwhile frequency of abnormal meiotic spindles increased with treatments.

Conclusions: Repetitive stimulations affected oocyte spindle morphology but did not induce changes in a set of proteins involved in cell cycle progression, usually altered in ovarian cancer. The significant increase of cyclin D1 in the FT requires further investigation.
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http://dx.doi.org/10.1007/s10815-014-0198-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048378PMC
June 2014

Post-ovulatory ageing of mouse oocytes affects the distribution of specific spindle-associated proteins and Akt expression levels.

Reprod Fertil Dev 2014 ;26(4):562-9

Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Embryology, School of Pharmacy and Medicine, 'Sapienza' University of Rome, V.le Regina Elena 324, 00161 Rome, Italy.

The aim of this study has been to determine the effects of in vivo post-ovulatory ageing (POA) on the distribution of spindle-associated proteins, histone H3/H4 post-translational modifications and on v-akt murine thymoma viral oncogene homolog 1 (Akt) expression levels. To this end, oocytes were retrieved 13, 29 and 33h after human chorionic gonadotrophin (hCG) treatment. The presence and distribution at the meiotic spindle of acetylated tubulin, γ-tubulin, polo kinase-1 and Ser473/Thr308 phosphorylated Akt (pAkt) as well as histone H3 and H4 acetylation and phosphorylation levels were assayed via immunofluorescence. Akt expression levels were determined via reverse transcription-polymerase chain reaction and western blotting analyses. Spindles from oocytes recovered 13h and 29h after hCG treatment showed similar levels of acetylated tubulin but ageing induced: (1) translocation of γ-tubulin from spindle poles to microtubules, (2) absence of Thr308- and Ser473-pAkt in 76% and 30% of oocytes, respectively, and (3) a significant reduction in phosphorylation levels of serine 10 on histone 3. At 29h, a significant decrease in Akt mRNA, but not in pAkt or Akt protein levels, was recorded. By contrast, protein content significantly decreased 33h after hCG. We conclude that POA impairs oocyte viability and fertilisability by altering the expression levels and spindle distribution of proteins that are implicated in cell survival and chromosome segregation. Together, these events could play a role in oocyte apoptosis.
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http://dx.doi.org/10.1071/RD13010DOI Listing
December 2014

[Multicentre, prospective cohort study, to validate the Italian version of the Braden Q scale for the risk of the pressure sores in newborns and up to 8 years old children].

Assist Inferm Ric 2012 Apr-Jun;31(2):83-90

Universita di Bologna.

Unlabelled: Multicenter prospective cohort study, to validate the Italian version of the Braden Q scale for the risk of pressure sores in newborns and up to 8 years old children.

Introduction: Children admitted to Intensive care Units (ICU), oncology and neurology/neurosurgery wards are at risk of developing pressure sores.

Aim: To validate the Italian version of the Braden Q scale for the assessment of the risk of developing pressure sores in children.

Methods: Children from 21 days to 8 years, admitted to intensive and sub intensive units were recruited. Premature babies, children admitted with a pressure sore and with a story of congenital cardiomiopathy were excluded. In this cohort, multicentre and with repeated measurements study, the first assessment was performed after 24 hours from hospital admission, using the Braden Q Scale (Suddaby's version). The pressure sores were assessed with the Skin assessment Tool and staged according to the National Pressure Ulcer Advisory Panel. RESULTS. On the 157 children 524 observation were conducted. The incidence of pressure sores was 17.2%. Only the analysis on specific subgroups of patients showed a good diagnostic accuracy: 71.4% on children 3-8 years; 85.6% in sub intensive wards.

Conclusions: The Braden Q scale may be reliably used and shows a good diagnostic accuracy in children 3-8 years of age admitted to sub-intensive, neurology, oncology and heamatology wards.
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http://dx.doi.org/10.1702/1131.12470DOI Listing
October 2012

The fungicide mancozeb induces toxic effects on mammalian granulosa cells.

Toxicol Appl Pharmacol 2012 Apr 17;260(2):155-61. Epub 2012 Feb 17.

Department of Health Sciences, University of L'Aquila, Via Vetoio, L'Aquila, Italy.

The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant.
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http://dx.doi.org/10.1016/j.taap.2012.02.005DOI Listing
April 2012

Effect of capacitation on the endocannabinoid system of mouse sperm.

Mol Cell Endocrinol 2011 Aug 25;343(1-2):88-92. Epub 2011 Jun 25.

Department of Biomedical Sciences, University of Teramo, Teramo 64100, Italy.

The presence of the elements of the endocannabinoid system (ECS) in sperm isolated from several species (from invertebrates to mammals, humans included) has supported the "evolutionary theory" that proposes endocannabinoids as check points in reproductive events like capacitation. In this study, we characterized the ECS elements at the mRNA, protein and functional levels in mouse sperm before and after capacitation. We found that the latter process increases the endogenous levels of the two major endocannabinoids (anandamide and 2-arachidonoylglycerol), through a decreased degradation and increased biosynthesis, respectively. Additionally, we found that the binding activity of cannabinoid receptors was not affected by sperm capacitation, whereas that of vanilloid receptor was reduced. Overall, our data demonstrate that mouse sperm have a fully functional ECS, and that capacitation alters the endogenous tone of the major endocannabinoids through distinct mechanisms.
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http://dx.doi.org/10.1016/j.mce.2011.01.022DOI Listing
August 2011

Effects of trifluralin on the mouse ovary.

Environ Toxicol 2013 Apr 4;28(4):201-6. Epub 2011 May 4.

Dipartimento di Scienze della Salute, Università degli Studi dell'Aquila, L'Aquila, Italy.

Trifluralin, a herbicide used to protect many arable and horticultural crops, was evaluated for its potential toxicity on the mammalian ovary. To this end, adult female mice were fed or not (control) with a trifluralin-enriched diet (150 mg/kg body weight/day) during gestation and lactation. After weaning, 3-week-old female mice from either trifluralin-treated or control groups were used to evaluate whether the exposure to this herbicide in utero and during lactation could induce stress responses in the ovary. It was found that trifluralin exposure caused a significantly higher level of p53, but not of pRb, in the whole ovary, and in particular in granulosa cells. TUNEL staining showed that herbicide treatment did not increase the apoptotic index of the somatic compartment. Also oocyte fertilizability was unaffected, as metaphase II oocytes retrieved from treated mice were capable of forming male and female pronuclei after in vitro fertilization as control mice. However, trifluralin determined a slightly higher number of oocytes with cytoplasmic degeneration compared with control animals. In conclusion, our results suggest that exposure to a low trifluralin dose during pregnancy and lactation does not impair oocyte quality, but can induce a stress response in ovarian somatic cells.
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http://dx.doi.org/10.1002/tox.20711DOI Listing
April 2013

Ultrastructure of isolated mouse ovarian follicles cultured in vitro.

Reprod Biol Endocrinol 2011 Jan 13;9. Epub 2011 Jan 13.

Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Rome, Italy.

Background: In vitro maturation of ovarian follicles, in combination with cryopreservation, might be a valuable method for preserving and/or restoring fertility in mammals with impaired reproductive function. Several culture systems capable of sustaining mammalian follicle growth in vitro have been developed and many studies exist on factors influencing the development of in vitro grown oocytes. However, a very few reports concern the ultrastructural morphology of in vitro grown follicles.

Methods: The present study was designed to evaluate, by transmission and scanning electron microscopy, the ultrastructural features of isolated mouse preantral follicles cultured in vitro for 6 days in a standard medium containing fetal calf serum (FCS). The culture was supplemented or not with FSH.

Results: The follicles cultured in FCS alone, without FSH supplementation (FCS follicles), did not form the antral cavity. They displayed low differentiation (juxta-nuclear aggregates of organelles in the ooplasm, a variable amount of microvilli on the oolemma, numerous granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). Eighty (80)% of FSH-treated follicles formed the antral cavity (FSH antral follicles). These follicles showed various ultrastructural markers of maturity (spreading of organelles in ooplasm, abundant microvilli on the oolemma, scarce granulosa cell-oolemma contacts, granulosa cell proliferation). Areas of detachment of the innermost granulosa cell layer from the oocyte were also found, along with a diffuse granulosa cell loosening compatible with the antral formation. Theca cells showed an immature morphology for the stage reached. Twenty (20)% of FSH-treated follicles did not develop the antral cavity (FSH non-antral follicles) and displayed morphological differentiation features intermediate between those shown by FCS and FSH antral follicles (spreading of organelles in the ooplasm, variable amount of microvilli, scattered granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment).

Conclusions: It is concluded that FSH supports the in vitro growth of follicles, but the presence of a diffuse structural granulosa cell-oocyte uncoupling and the absence of theca development unveil the incomplete efficiency of the system. The present study contributes to explain, from a morphological point of view, the effects of culture conditions on the development of mouse in vitro grown follicles and to highlight the necessity of maintaining efficient intercellular communications to obtain large numbers of fully-grown mature germ cells.
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http://dx.doi.org/10.1186/1477-7827-9-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033320PMC
January 2011

Akt expression in mouse oocytes matured in vivo and in vitro.

Reprod Biomed Online 2010 Jan 30;20(1):35-41. Epub 2009 Oct 30.

Department of Health Sciences, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy.

To improve developmental competence of in vitro matured oocytes, culture medium can be supplemented with hypoxanthine (Hx) and FSH or epidermal growth factor (EGF) to trigger the activation of essential signalling pathways regulating meiotic resumption and progression. Since the serine/threonine kinase, Akt, contributes to the regulation of the meiotic cell cycle, this study analysed its expression level and localization at the meiotic spindle in oocytes matured in vivo or in vitro in the presence of Hx-FSH or Hx-EGF. Independently of culture conditions adopted, Akt mRNA concentration did not vary from germinal vesicle to metaphase I (MI), while at MII a significant decrease in Akt1 mRNA concentration was recorded in oocytes matured in vivo and in those stimulated by Hx-EGF (P < 0.05). Phoshorylated Akt protein content was similar in the different groups of MI oocytes, but it decreased at MII in oocytes matured either in vivo or in vitro with Hx-EGF. Ser-473-phosphorylated Akt was localized uniformly to the meiotic spindle in more than 90% of oocytes. These results indicate that, in mouse oocytes, Akt expression is differentially regulated during in vivo and in vitro maturation and suggest that EGF could be a positive modulator, even stronger than FSH, of oocyte meiotic maturation.
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http://dx.doi.org/10.1016/j.rbmo.2009.10.011DOI Listing
January 2010

Modulation of the endocannabinoid-degrading enzyme fatty acid amide hydrolase by follicle-stimulating hormone.

Vitam Horm 2009 ;81:231-61

Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, Italy.

Follicle-stimulating hormone (FSH) is a glycoprotein that transmits its signals via a G protein-coupled receptor. As yet, not many targets of FSH have been identified, able to justify the critical role of this hormone on reproductive events. On the other hand, among the biological activities of the endocannabinoid anandamide (AEA), growing interest has been attracted by the regulation of mammalian fertility. Recently, we have shown that treatment of mouse primary Sertoli cells with FSH enhances the activity of the AEA hydrolase (fatty acid amide hydrolase, FAAH), whereas it does not affect the enzymes that synthesize AEA, nor the level of the AEA-binding type-2 cannabinoid and type-1 vanilloid receptors. In addition, diacylglycerol lipase and monoacylglycerol lipase, which, respectively, synthesize and degrade the other major endocannabinoid 2-arachidonoylglycerol, were not regulated by FSH. Interestingly, FAAH stimulation by FSH occurred through protein kinase A and aromatase-dependent pathways that were able to modulate FAAH activity (via phosphorylation of accessory proteins) and faah gene expression (via an estrogen response element on the promoter region). Taken together, these data identify FAAH as the only target of FSH among the elements of the endocannabinoid system, with a critical impact on Sertoli cell proliferation, and thus spermatogenesis and male reproduction.
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http://dx.doi.org/10.1016/S0083-6729(09)81010-8DOI Listing
September 2009

The effects of the endocrine disruptors dithiocarbamates on the mammalian ovary with particular regard to mancozeb.

Curr Pharm Des 2007 ;13(29):2989-3004

Department of Biological Sciences and Technologies, Centre of Electron Microscope, University of L'Aquila, L'Aquila, Italy.

Many human-made chemicals are called endocrine disruptors (EDs) because they have the potential to disrupt endocrine functions in exposed organisms. Many EDs can disrupt hormonal homeostasis by interfering with hormone receptor recognition, binding and activation, while others act by still unknown mechanisms. Among the EDs specifically affecting the female reproductive system, those with steroidogenic/antisteroidogenic effects have been extensively studied and the mechanisms of toxicity clarified also at molecular level. For many others, information is restricted to few epidemiological data and in vivo/in vitro experiments with animal models. This is the case of the dithiocarbamates, and in particular of the fungicide mancozeb, an ethylenedithiocarbamate widely used to protect fruit and vegetables, ginseng included, because of its low acute toxicity in humans. Although the mechanism(s) by which mancozeb may specifically act on female reproductive organs are largely unknown, data on experimental animals in vivo have demonstrated that the fungicide can induce several disturbances on estrus cycle. When used in vitro at concentrations considered too low to cause human health injuries, the fungicide impairs mouse embryo development and meiotic spindle assembly. The possibility that the female germ cell (the oocyte) could be a specific target of mancozeb suggests a role for this fungicide as probable inductor of infertility also in exposed human populations.
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http://dx.doi.org/10.2174/138161207782110516DOI Listing
January 2008

Follicle-stimulating hormone activates fatty acid amide hydrolase by protein kinase A and aromatase-dependent pathways in mouse primary Sertoli cells.

Endocrinology 2007 Mar 16;148(3):1431-9. Epub 2006 Nov 16.

Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy.

Among the biological activities of the endocannabinoid anandamide (N-arachidonoylethanolamine) (AEA), growing interest has been attracted by the regulation of mammalian fertility. Recently we have shown that treatment of mouse primary Sertoli cells with FSH enhances the activity of the AEA hydrolase [fatty acid amide hydrolase (FAAH)], though the molecular details were not elucidated. Here, we investigated whether FSH was also able to affect the enzymes that synthesize AEA (N-acyltransferase and N-acyl-phosphatidyl-ethanolamine-phospholipase D), the endogenous content of this endocannabinoid, and the level of the AEA-binding vanilloid receptor 1 (transient receptor potential channel vanilloid receptor subunit 1). We show that FSH enhanced FAAH activity (up to approximately 500% of the controls) and expression (up to approximately 300%), leading to a marked reduction (down to approximately 15%) of AEA content. However N-acyltransferase and N-acyl-phosphatidyl-ethanolamine-phospholipase D activity, and transient receptor potential channel vanilloid receptor subunit 1 binding were not affected. We also show that diacylglycerol lipase and monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoyl-glycerol, were not regulated by FSH, neither was the membrane transport of this endocannabinoid. In addition, we show that FAAH stimulation by FSH was abrogated by inhibitors of protein kinase A (PKA) and cytochrome-P(450) aromatase, and was conversely mimicked by N,O'-dibutyryl cAMP and estrogen. Finally, we demonstrate that FSH protects Sertoli cells against the pro-apoptotic activity of AEA, through PKA and aromatase-dependent activation of FAAH. Altogether these data suggest that FAAH is the only target of FSH among the elements of the endocannabinoid system, and that its regulation by PKA and aromatase-dependent pathways impacts Sertoli cell proliferation.
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http://dx.doi.org/10.1210/en.2006-0969DOI Listing
March 2007

Mouse oocyte differentiation during antral follicle development.

Microsc Res Tech 2006 Jun;69(6):408-14

Department of Biomedical Sciences and Technologies, University of L'Aquila, L'Aquila, Italy.

During antral follicle development mouse oocytes undergo rearrangement of granulosa cell interactions and the oocytes released from follicles at the beginning or at the end of antral development are either devoid of denuded oocytes (DO) or strictly associated with cumulus-intact (CI) cumulus cells. In this study, these two oocyte classes were analyzed before germinal vesicle (GV) and after in vitro maturation (IVM) to evaluate (a) the ultrastructural aspect of oolemma microvilli by scanning electron microscopy analysis and (b) specific morphological markers of differentiation (chromatin organization, mitochondria, cortical granules, microfilaments, and spindle of metaphase II- MII-). At GV-stage, CI oocytes exhibited remarkable differences (a) in the oolemma microvillar ultrastructure and distribution with respect to DO and (b) in the chromatin organization that was typical of meiotically competent germ cells. By contrast, homogeneous patterns of distribution of mitochondria, cortical granules, and microfilaments characterized both the oocyte classes. At the end of culture, CI oocytes, even when matured without cumulus cells, reached more efficiently the MII stage and acquired an ultrastructural microvillous configuration different from DO. In addition, MII-arrested DO had a higher percentage of meiotic spindles with abnormal morphology in comparison with preovulatory oocytes, while cortical granule and microfilament patterns revealed no appreciable differences between the groups. With regard to mitochondria, a polarized distribution of these organelles was found in 82% of DO and in 97% of CI oocytes. These observations suggested that the achievement of the full antral follicle development is a condition for the acquisition of specific qualitative properties that are essential for the production of fertilizable oocytes, both in in vivo and in vitro models as well.
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http://dx.doi.org/10.1002/jemt.20300DOI Listing
June 2006

Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes.

Reprod Toxicol 2006 Jul 10;22(1):51-5. Epub 2006 Jan 10.

Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di L'Aquila, 67100 L'Aquila, Italy.

In this study the effects of mancozeb, a widely used ethylenebisdithiocarbamate fungicide, on mouse oocyte meiotic maturation and fertilization were analyzed. Oocyte cumulus cell-complexes were matured in vitro with or without increasing concentrations of the fungicide (from 0.001 to 1 microg/ml) that, due to its different stability in organic solvents and in water, was resuspended either in dimethyl sulfoxide or in culture medium. Although, about 95% of oocytes reached the metaphase II stage; mancozeb-exposed oocytes showed a dose-dependent increase of alterations in spindle morphology, and this negative effect was more evident when the fungicide was resuspended in culture medium. Under the latter culture condition, oocytes matured in the presence of 0.1 and 1 microg/ml mancozeb showed a significant reduction also in the formation of male and female pronuclei. These results indicate that mancozeb can adversely affect mammalian reproductive performance, likely by perturbing microtubular organization during meiotic maturation.
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http://dx.doi.org/10.1016/j.reprotox.2005.11.005DOI Listing
July 2006

Mancozeb exposure in vivo impairs mouse oocyte fertilizability.

Reprod Toxicol 2006 Feb 4;21(2):216-9. Epub 2005 Oct 4.

Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di L'Aquila, 67100 L'Aquila, Italy.

Mancozeb is known to alter reproductive performance in exposed animals, but its specific mechanism of action is still unclear. We investigated whether in female mice of the F1 generation, mancozeb could affect oocyte ability to undergo complete meiotic maturation and fertilization. Female mice were treated with 50 and 500 mg/kg of mancozeb (or vehicle in the controls) from gestational day 2 to postnatal day 20. Results demonstrated that only at the highest dose, mancozeb induced a significant decrease in the number of ovulated eggs. Moreover, at this dose mancozeb caused a significant decrease of fertilizability related to a reduction of the formation of male and female pronuclei.
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http://dx.doi.org/10.1016/j.reprotox.2005.08.004DOI Listing
February 2006

Influence of thyroid hormone on mouse preantral follicle development in vitro.

Fertil Steril 2004 Mar;81 Suppl 1:919-24

Dipartimento di Scienze e Tecnologie Biomediche-L'Aquila, Via Vetoio 10, 67100 L'Aquila, Italy.

Objective: To evaluate the role of 3,3',5-triiodothyronine (T(3)) on ovarian follicle development in vitro.

Design: Experimental study.

Setting: University reproductive biology unit.

Animal(s): CD1 mice (female).

Intervention(s): In vitro culture.

Main Outcome Measure(s): Assessment of follicle and oocyte development following a 6-day culture with FSH (100 mU/mL), (Bu)(2)cAMP (0.5 mM), and T(3) (1-100 nM), alone or in combination.

Result(s): A high percentage of in vitro grown (IVG) FSH-stimulated follicles formed an antral cavity (AC), while the addition of T(3) (100 nM) significantly reduced this response in a statistically significant manner. A statistically significant recovery of AC formation was obtained by limiting exposure to 4 days of culture. Formation of AC was induced by (Bu)(2)cAMP, which prevented T(3)-mediated suppression of AC formation. Under different conditions, high proportions of germinal vesicle-arrested IVG oocytes displayed condensed chromatin configuration. The capacity to undergo germinal vesicle breakdown (GVBD) was similar in all groups, and a statistically significant reduction in the percentage of oocytes with PB1 was recorded when T(3) was added to FSH or (Bu)(2)cAMP. This ability was partially recovered by removing T(3) on day 4 of culture.

Conclusion(s): Exposure to high T(3) concentrations can impair preantral follicle development, but this effect can be partly reverted by restoring a physiological hormonal milieu before follicle commitment to antral development.
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http://dx.doi.org/10.1016/j.fertnstert.2003.11.014DOI Listing
March 2004

Effect of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide on mouse preantral follicle development in vitro.

Endocrinology 2004 Apr 30;145(4):2071-9. Epub 2003 Dec 30.

Department of Biomedical Sciences and Technologies, Faculty of Medicine, University of L'Aquila, Italy.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide isolated from ovine hypothalamus. It is transiently expressed in preovulatory follicles and positively affects several parameters correlated with the ovulatory process. It has also been shown to be expressed in the interstitial tissue around primordial and preantral follicles. The aim of the present study was to investigate whether PACAP influences preantral follicle growth and differentiation. Mouse preantral follicles were cultured for 5 d in the presence of FSH and increasing concentrations of PACAP or vasoactive intestinal polypeptide (VIP) (10(-12) to 10(-7) m). In the presence of FSH, follicles increased in diameter and formed an antrum. At the concentrations tested, neither PACAP alone nor VIP alone had any effect on follicle development, but the addition of either peptide to FSH-stimulated follicles caused a dose-dependent inhibition of follicle growth, antrum formation, granulosa cell proliferation, and estradiol production. The effect of PACAP on follicle growth and antrum formation was directly correlated with the length of stimulation and was reversible. Although exposure of follicles to 10(-7) m PACAP and VIP did not affect oocyte growth, it severely impaired completion of meiotic maturation in oocytes isolated from the follicles and cultured for 17 h in medium alone. The cyclic production of PACAP by preovulatory follicles during the estrous cycle in adult rats and its induction by LH in the rat and mouse ovary suggest that this peptide may play a role in the local regulation of preantral follicle growth.
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http://dx.doi.org/10.1210/en.2003-1004DOI Listing
April 2004

Anandamide activity and degradation are regulated by early postnatal aging and follicle-stimulating hormone in mouse Sertoli cells.

Endocrinology 2003 Jan;144(1):20-8

Department of Experimental Medicine and Biochemical Sciences, University of Rome, Tor Vergata, I-00133 Italy.

Anandamide (AEA), a prominent member of the endogenous ligands of cannabinoid receptors (endocannabinoids), is known to adversely affect female fertility. However, a potential role of AEA in male reproductive functions is unknown. Here we report evidence that immature mouse Sertoli cells have the biochemical tools to bind and inactivate AEA, i.e. a functional type-2 cannabinoid receptor (CB2R), a selective AEA membrane transporter, and an AEA-degrading enzyme fatty acid amide hydrolase. We show that, unlike CB2R, the activity of AEA membrane transporter and the activity and expression of FAAH decrease, whereas the apoptosis-inducing activity of AEA increases with age during the neonatal period. We also show that FSH reduces the apoptotic potential of AEA, but not that of its nonhydrolyzable analog methanandamide. Concomitantly, FSH enhances FAAH activity in a manner dependent on mRNA transcription and protein synthesis and apparently involving cAMP. These data demonstrate that Sertoli cells partake in the peripheral endocannabinoid system, and that FSH reduces the apoptotic potential of AEA by activating FAAH. Taken together, it can be suggested that the endocannabinoid network plays a role in the hormonal regulation of male fertility.
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http://dx.doi.org/10.1210/en.2002-220544DOI Listing
January 2003