Publications by authors named "Rita Canipari"

30 Publications

  • Page 1 of 1

Female Fertility and Environmental Pollution.

Int J Environ Res Public Health 2020 11 26;17(23). Epub 2020 Nov 26.

Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze della Vita e dell'Ambiente, Università degli Studi dell'Aquila, 67100 L'Aquila, Italy.

A realistic picture of our world shows that it is heavily polluted everywhere. Coastal regions and oceans are polluted by farm fertilizer, manure runoff, sewage and industrial discharges, and large isles of waste plastic are floating around, impacting sea life. Terrestrial ecosystems are contaminated by heavy metals and organic chemicals that can be taken up by and accumulate in crop plants, and water tables are heavily contaminated by untreated industrial discharges. As deadly particulates can drift far, poor air quality has become a significant global problem and one that is not exclusive to major industrialized cities. The consequences are a dramatic impairment of our ecosystem and biodiversity and increases in degenerative or man-made diseases. In this respect, it has been demonstrated that environmental pollution impairs fertility in all mammalian species. The worst consequences are observed for females since the number of germ cells present in the ovary is fixed during fetal life, and the cells are not renewable. This means that any pollutant affecting hormonal homeostasis and/or the reproductive apparatus inevitably harms reproductive performance. This decline will have important social and economic consequences that can no longer be overlooked.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijerph17238802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730072PMC
November 2020

Loss of Two-Pore Channel 2 (TPC2) Expression Increases the Metastatic Traits of Melanoma Cells by a Mechanism Involving the Hippo Signalling Pathway and Store-Operated Calcium Entry.

Cancers (Basel) 2020 Aug 24;12(9). Epub 2020 Aug 24.

Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, SAPIENZA University of Rome, 16 Via A. Scarpa, 00161 Roma, Italy.

Melanoma is one of the most aggressive and treatment-resistant human cancers. The two-pore channel 2 (TPC2) is located on late endosomes, lysosomes and melanosomes. Here, we characterized how TPC2 knockout (KO) affected human melanoma cells derived from a metastatic site. TPC2 KO increased these cells' ability to invade the extracelullar matrix and was associated with the increased expression of mesenchymal markers ZEB-1, Vimentin and N-Cadherin, and the enhanced secretion of MMP9. TPC2 KO also activated genes regulated by YAP/TAZ, which are key regulators of tumourigenesis and metastasis. Expression levels of ORAI1, a component of store-operated Ca entry (SOCE), and PKC-βII, part of the HIPPO pathway that negatively regulates YAP/TAZ activity, were reduced by TPC2 KO and RNA interference knockdown. We propose a cellular mechanism mediated by ORAI1/Ca/PKC-βII to explain these findings. Highlighting their potential clinical significance, patients with metastatic tumours showed a reduction in TPC2 expression. Our research indicates a novel role of TPC2 in melanoma. While TPC2 loss may not activate YAP/TAZ target genes in primary melanoma, in metastatic melanoma it could activate such genes and increase cancer aggressiveness. These findings aid the understanding of tumourigenesis mechanisms and could provide new diagnostic and treatment strategies for skin cancer and other metastatic cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12092391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564716PMC
August 2020

Discovery of the First Human Arylsulfatase A Reversible Inhibitor Impairing Mouse Oocyte Fertilization.

ACS Chem Biol 2020 06 14;15(6):1349-1357. Epub 2020 Apr 14.

Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy.

Arylsulfatase A (ARSA) plays a crucial role in the reproduction of mammals due to its involvement in the specific gamete interaction preceding sperm and egg fusion leading to fertilization. Recently, it has been shown that zona pellucida (ZP) sperm binding and fertilization in mice are markedly hampered by using a specific anti-ARSA antibody. Herein, the design and discovery of the first ARSA small molecule inhibitor based on a coumarin-containing polycycle are presented. Through a structure-based approach applied on our in-house library, compound was identified as an ARSA reversible inhibitor (ARSAi); then its activity was validated through both surface plasmon resonance and biochemical inhibition experiments, the first providing a value of 21 μM and the latter an IC value of 13.2 μM. Further investigations highlighted that compound induced 20% sperm death at 25 μM and also impaired sperm motility; nevertheless both the effects were mediated by ROS production, since they were rescued by the cotreatment of and -acetyl cysteine (NAC). Interestingly, while was not able to hamper the ZP/sperm binding, it markedly decreased the oocyte fertilization by mouse sperm up to 60%. Notably, this effect was not hampered by /NAC coadministration, hence allowing the ruling out of an ROS-dependent mechanism. In conclusion, herein is reported the first ever hit of ARSAi as a chemical tool that will enable better exploration of ARSA's biological role in fertilization as well as provide a starting point for developing structure optimization aimed at increasing enzyme inhibition potency but also providing a deeper understanding of the involvement of ARSA in the fertilization pathway mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.9b00999DOI Listing
June 2020

Incidence, Origin, and Predictive Model for the Detection and Clinical Management of Segmental Aneuploidies in Human Embryos.

Am J Hum Genet 2020 04 26;106(4):525-534. Epub 2020 Mar 26.

Igenomix Italy, 36063, Marostica, Italy; DAHFMO, Unit of Histology and Medical Embryology, Sapienza, University of Rome, 00161, Roma, Italy; Igenomix Foundation, 46980, Valencia, Spain. Electronic address:

Despite next-generation sequencing, which now allows for the accurate detection of segmental aneuploidies from in vitro fertilization embryo biopsies, the origin and characteristics of these aneuploidies are still relatively unknown. Using a multifocal biopsy approach (four trophectoderms [TEs] and one inner cell mass [ICM] analyzed per blastocyst; n = 390), we determine the origin of the aneuploidy and the diagnostic predictive value of segmental aneuploidy detection in TE biopsies toward the ICM's chromosomal constitution. Contrary to the prevalent meiotic origin of whole-chromosome aneuploidies, we show that sub-chromosomal abnormalities in human blastocysts arise from mitotic errors in around 70% of cases. As a consequence, the positive-predictive value toward ICM configuration was significantly lower for segmental as compared to whole-chromosome aneuploidies (70.8% versus 97.18%, respectively). In order to enhance the clinical utility of reporting segmental findings in clinical TE biopsies, we have developed and clinically verified a risk stratification model based on a second TE biopsy confirmation and segmental length; this model can significantly improve the prediction of aneuploidy risk in the ICM in over 86% of clinical cases enrolled. In conclusion, we provide evidence of the predominant mitotic origin of segmental aneuploidies in preimplantation embryos and develop a risk stratification model that can help post-test genetic counseling and that facilitates the decision-making process on clinical utilization of these embryos.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2020.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118577PMC
April 2020

Definition and validation of a custom protocol to detect miRNAs in the spent media after blastocyst culture: searching for biomarkers of implantation.

Hum Reprod 2019 09;34(9):1746-1761

DAHFMO, Unit of Histology and Medical Embryology, Sapienza, University of Rome, Italy.

Study Question: Can miRNAs be reliably detected in the spent blastocyst media (SBM) after IVF as putative biomarkers of the implantation potential of euploid embryos?

Summary Answer: Adjustment of the data for blastocyst quality and the day of full-expansion hinders the predictive power of a fast, inexpensive, reproducible and user-friendly protocol based on the detection of 10 selected miRNAs from SBM.

What Is Known Already: Euploidy represents so far the strongest predictor of blastocyst competence. Nevertheless, ~50% of the euploid blastocysts fail to implant. Several studies across the years have suggested that a dialogue exists between the embryo and the endometrium aimed at the establishment of a pregnancy. MicroRNAs have been proposed as mediators of such a dialogue and investigated in this respect. Several expensive, time-consuming and complex protocols have been adopted and promising results have been produced, but conclusive evidence from large clinical studies is missing.

Study Design, Size, Duration: This study was conducted in two phases from September 2015 to December 2017. In Phase 1, the human blastocyst miRNome profile was defined from the inner cell mass (ICM) and the corresponding whole-trophectoderm (TE) of six donated blastocysts. Two different protocols were adopted to this end. In parallel, 6 pools of 10 SBM each were run (3 from only implanted euploid blastocysts, IEBs; and 3 from only not-implanted euploid blastocysts, not-IEBs). A fast, inexpensive and user-friendly custom protocol for miRNA SBM profiling was designed. In Phase 2, 239 SBM from IEB and not-IEB were collected at three IVF centres. After 18 SBM from poor-quality blastocysts were excluded from the analysis, data from 107 SBM from not-IEB and 114 from IEB were produced through the previously developed custom protocol and compared. The data were corrected through logistic regressions.

Participant/materials, Settings, Methods: Donated blastocysts underwent ICM and whole-TE isolation. SBM were collected during IVF cycles characterized by ICSI, blastocyst culture in a continuous media, TE biopsy without zona pellucida opening in Day 3, quantitative PCR (qPCR)-based aneuploidy testing and vitrified-warmed single euploid embryo transfer. Not-IEB and IEB were clustered following a negative pregnancy test and a live birth, respectively. The Taqman Low Density Array (TLDA) cards and the Exiqon microRNA human panel I+II qPCR analysis protocols were adopted to analyse the ICM and whole-TE. The latter was used also for SBM pools. A custom protocol and plate was then designed based on the Exiqon workflow, validated and finally adopted for SBM analysis in study Phase 2. This custom protocol allows the analysis of 10 miRNAs from 10 SBM in 3 hours from sample collection to data inspection.

Main Results And Role Of The Chance: The TLDA cards protocol involved a higher rate of false positive results (5.6% versus 2.8% with Exiqon). There were 44 miRNAs detected in the ICM and TE from both the protocols. One and 24 miRNAs were instead detected solely in the ICM and the TE, respectively. Overall, 29 miRNAs were detected in the pooled SBM: 8 only from not-IEB, 8 only from IEB and 13 from both. Most of them (N = 24/29, 82.7%) were also detected previously in both the ICM and TE with the Exiqon protocol; two miRNAs (N = 2/29, 6.9%) were previously detected only in the TE, and three (N = 3/29, 10.3%) were never detected previously. In study Phase 2, significant differences were shown between not-IEB and IEB in terms of both miRNA detection and relative quantitation. However, when the data were corrected for embryo morphology and day of full development (i.e. SBM collection), no significant association was confirmed.

Limitations, Reasons For Caution: This study did not evaluate specifically exosomal miRNAs, thereby reducing the chance of identifying the functional miRNAs. Ex-vivo experiments are required to confirm the role of miRNAs in mediating the dialogue with endometrial cells, and higher throughput technologies need to be further evaluated for miRNA profiling from clinical SBM samples.

Wider Implications Of The Findings: Although no clinical predictive power was reported in this study, the absence of invasiveness related with SBM analysis and the evidence that embryonic genetic material can be reliably detected and analysed from SBM make this waste product of IVF an important source for further investigations aimed at improving embryo selection.

Study Funding/competing Interest(s): This project has been financially supported by Merck KgaA (Darmstadt, Germany) with a Grant for Fertility Innovation (GFI) 2015. The authors have no conflict of interest to declare related with this study.

Trial Registration Number: None.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/humrep/dez119DOI Listing
September 2019

Stem-like and highly invasive prostate cancer cells expressing CD44v8-10 marker originate from CD44-negative cells.

Oncotarget 2018 Jul 20;9(56):30905-30918. Epub 2018 Jul 20.

Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Section of Histology and Medical Embryology, Sapienza University, Rome, Italy.

In human prostate cancer (PCa), the neuroendocrine cells, expressing the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. During the study of heterogeneity of the highly aggressive neuroendocrine PCa cell lines PC3 and DU-145, we isolated and expanded a minor subpopulation of very small cells lacking CD44 (CD44). Unexpectedly, these sorted CD44 cells rapidly and spontaneously converted to a stable CD44 phenotype specifically expressing the CD44v8-10 isoform which the sorted CD44 subpopulation failed to express. Surprisingly and potentially interesting, in these cells expression of CD44v8-10 was found to be induced in stem cell medium. CD44 variant isoforms are known to be more expressed in CSC and metastatic cells than CD44 standard isoform. In agreement, functional analysis of the two sorted and cultured subpopulations has shown that the CD44v8-10 PC3 cells, resulting from the conversion of the CD44 subpopulation, were more invasive and had a higher clonogenic potential than the sorted CD44 cells, in that they produced mainly holoclones, known to be enriched in stem-like cells. Of interest, the CD44v8-10 is more expressed in human PCa biopsies than in normal gland. The discovery of CD44v8-10 cells with stem-like and invasive features, derived from a minoritarian CD44 cell population in PCa, alerts on the high plasticity of stem-like markers and urges for prudency on the approaches to targeting the putative CSC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.25773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6089404PMC
July 2018

Effect of mitotane on mouse ovarian follicle development and fertility.

J Endocrinol 2017 Jul 27;234(1):29-39. Epub 2017 Apr 27.

EndocrinologyDepartment of Clinical and Molecular Medicine, Sant'Andrea Hospital, Sapienza University of Rome, Rome, Italy.

Mitotane (MTT) is an adrenolytic drug used in advanced and adjuvant treatment of adrenocortical carcinoma, in Cushing's disease and in ectopic syndrome. However, knowledge about its effects on the ovary is still scarce. The purpose of this study is to investigate the effect of MTT on the ovary using and models. The study was performed in CD1 mice and in the COV-434 human ovarian granulosa cell line. We examined ovarian morphology, follicle development, steroidogenesis and procreative function in mice and the effect of MTT on cell growth Our results revealed that treatment of CD1 mice with MTT induces a decrease in early antral follicles with a subsequent increase in the secondary follicles, measured by the increased levels of anti-Mullerian Hormone ( < 0.05) and decreased levels of FSH receptor ( < 0.05). Moreover, we observed a significant decrease in ( < 0.01) and ( < 0.001) mRNA level in MTT-treated animals. Ovulation, induced by PMSG/hCG stimulation, was also significantly impaired, with a reduction in the number of ovulated oocytes ( < 0.01) and fewer corpora lutea in treated animals. Likewise, the mating experiment demonstrated a delay in the time of conception as well as fewer pups per litter in MTT-treated mice ( < 0.05). Experiments performed on the COV-434 cell line showed a significant inhibition of growth followed by apoptosis ( < 0.01). In conclusion, our study highlights the key points of ovarian folliculogenesis affected by MTT and demonstrates impairment of the ovulation process with a negative impact on conception, which is nevertheless preserved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/JOE-17-0203DOI Listing
July 2017

The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis.

Biomed Res Int 2016 28;2016:7193075. Epub 2016 Jan 28.

GENERA Centre for Reproductive Medicine, Clinica Valle Giulia, Via G. de Notaris 2/b, 00197 Rome, Italy; GENETYX, Molecular Biology Laboratory, Via Fermi 1, 36063 Marostica, Italy.

Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2016/7193075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749789PMC
November 2016

Paradoxical E-cadherin increase in 5FU-resistant colon cancer is unaffected during mesenchymal-epithelial reversion induced by γ-secretase inhibition.

Life Sci 2016 Jan 30;145:174-83. Epub 2015 Dec 30.

Department of Experimental Medicine, Sapienza University of Rome, Systems Biology Group Lab, viale Regina Elena 324, 00161 Rome, Italy; Systems Biology Group Lab, Sapienza University of Rome, Rome, Italy. Electronic address:

Aim: Presenilin-1 (PS1), the main component of γ-secretase activity support a key role during Epithelial-Mesenchymal Transition (EMT) and chemoresistance acquisition by triggering a complex sequence of molecular events, including E-cadherin down-regulation. However, we hypothesize that EMT and chemoresistance should be deemed separate processes in HCT-8 colon cancer cells.

Main Methods: HCT-8 and HCT-8FUres invasion was evaluated by trans-well assay. uPA activity was detected by zymography. Prostaglandin E2 levels were quantified using an ELISA kit. E-cadherin FL and CTF2, PS1, Notch1, Cyclin D1, COX2, SNAI1 and α-SMA expression were determined using Western blot technique. β-Catenin localization was observed by confocal microscopy. Cell apoptosis was evaluated by cytofluorimetric assay, and measurement of caspase-3 and cl-PARP. γ-Secretase activity was inhibited by DAPT, a γ-secretase inhibitor.

Key Findings: Chemoresistant HCT-8 underwent EMT that can be efficiently reversed by inhibiting PS1 activity, leading thus to a normalization of mostly of the pivotal features showed by the invasive cancer phenotype. Indeed, we observed decreased SNAI1 and Notch 1 activation, altogether with reduced E-cadherin cleavage. Concomitantly, resistant HCT-8 invasiveness was almost completely abolished. However, such reversion was not followed by any increase in apoptotic rate, not by changes in E-cadherin levels. Indeed, despite HCT-8FUres underwent an undeniable EMT, full-length E-cadherin levels were found remarkably higher than those observed in wild HCT-8.

Significance: High E-cadherin concentration in presence of enhanced γ-secretase activity is incontestably a paradoxically result, highlighting that E-cadherin loss is not a pre-requisite for EMT. Additionally, EMT and chemoresistance acquisition in HCT-8 should be considered as distinct processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.lfs.2015.12.048DOI Listing
January 2016

The Plasminogen System and Transforming Growth Factor-β in Subjects With Obstructive Sleep Apnea Syndrome: Effects of CPAP Treatment.

Respir Care 2015 Nov 18;60(11):1643-51. Epub 2015 Aug 18.

Section of Histology, Department of Anatomy, Histology, Forensic Medicine, and Orthopedics, Sapienza University of Rome, Via A Scarpa 16, Rome, Italy.

Background: Obstructive sleep apnea syndrome (OSAS) has emerged as a risk factor for cardiovascular disease. A prothrombotic state may affect coagulation and participate in the atherosclerotic process in subjects with OSAS. These alterations in coagulation seem to involve the plasminogen activation system. We evaluated the imbalances of the plasminogen activation system related to OSAS, and we assessed the effects of CPAP on the plasminogen activation system.

Methods: Thirty-nine subjects were submitted to a home-based cardiorespiratory sleep study, and 14 healthy subjects (apnea-hypopnea index < 5) were used as controls. Serum levels of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and active transforming growth factor-β (TGF-β) were measured. These molecules were reassessed in only 17 of the subjects after 1 month of CPAP.

Results: PAI-1 and tPA were significantly higher in the subjects with OSAS compared with the controls, whereas TGF-β and uPA levels were lower. PAI-1 showed a significant positive correlation with the apnea-hypopnea index, percentage of time spent at O2 saturation < 90%, and oxygen desaturation index, whereas TGF-β was inversely related to all 3 of these parameters. After the CPAP therapy, PAI-1 significantly decreased, whereas TGF-β showed a significant increase, although the values did not reach those of the controls. uPA and tPA did not show significant differences after the treatment.

Conclusions: Our results suggest an imbalance of fibrinolysis related to OSAS and an improvement of the prothrombotic state after the CPAP treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4187/respcare.03571DOI Listing
November 2015

HGF Modulates Actin Cytoskeleton Remodeling and Contraction in Testicular Myoid Cells.

Biomedicines 2015 Jan 28;3(1):89-109. Epub 2015 Jan 28.

Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Embryology, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy.

The presence of the HGF/Met system in the testicular myoid cells was first discovered by our group. However, the physiological role of this pathway remains poorly understood. We previously reported that HGF increases uPA secretion and TGF-β activation in cultured tubular fragments and that HGF is maximally expressed at Stages VII-VIII of the seminiferous epithelium cycle, when myoid cell contraction occurs. It is well known that the HGF/Met pathway is involved in cytoskeletal remodeling; moreover, the interaction of uPA with its receptor, uPAR, as well as the activation of TGF-β have been reported to be related to the actin cytoskeleton contractility of smooth muscle cells. Herein, we report that HGF induces actin cytoskeleton remodeling in isolated myoid cells and myoid cell contraction in cultured seminiferous tubules. To better understand these phenomena, we evaluated: (1) the regulation of the uPA machinery in isolated myoid cells after HGF administration; and (2) the effect of uPA or Met inhibition on HGF-treated tubular fragments. Because uPA activates latent TGF-β, the secretion of this factor was also evaluated. We found that both uPA and TGF-β activation increase after HGF administration. In testicular tubular fragments, HGF-induced TGF-β activation and myoid cell contraction are abrogated by uPA or Met inhibitor administration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biomedicines3010089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5344232PMC
January 2015

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10815-014-0198-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048378PMC
June 2014

Grape seed extract suppresses MDA-MB231 breast cancer cell migration and invasion.

Eur J Nutr 2014 11;53(2):421-31. Epub 2013 Jun 11.

Department of Clinical and Molecular Medicine, La Sapienza University, Piazza Sassari 3, 00161, Rome, Italy.

Background And Aim: Breast cancer remains a leading cause of mortality among women. In metastasis, cascade migration of cancer cells and invasion of extracellular matrix (ECM) represent critical steps. Urokinase-type plasminogen activator (uPA), as well as metalloproteinases MMP-2 and MMP-9, strongly contribute to ECM remodelling, thus becoming associated with tumour migration and invasion. In addition, the high expression of cytoskeletal (CSK) proteins, as fascin, has been correlated with clinically aggressive metastatic tumours, and CSK proteins are thought to affect the migration of cancer cells. Consumption of fruits and vegetables, characterized by high procyanidin content, has been associated to a reduced mortality for breast cancer. Therefore, we investigated the biological effect of grape seed extract (GSE) on the highly metastatic MDA-MB231 breast cancer cell line, focusing on studying GSE ability in inhibiting two main metastatic processes, i.e., cell migration and invasion.

Methods: After MDA-MB231 breast cancer cells stimulated with GSE migration and invasion were evaluated by means of trans-well assays and uPA as well as MMPs activity was detected by gelatin zymography. Fascin, β-catenin and nuclear factor-κB (NF-κB) expression were determined using western blot technique. β-Catenin localization was observed by confocal microscopy.

Results: We observed that high concentrations of GSE inhibited cell proliferation and apoptosis. Conversely, low GSE concentration decreased cell migration and invasion, likely by hampering β-catenin expression and localization, fascin and NF-κB expression, as well as by decreasing the activity of uPA, MMP-2 and MMP-9.

Conclusions: These results make GSE a powerful candidate for developing preventive agents against cancer metastasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00394-013-0542-6DOI Listing
October 2014

Expression and functional activity of PACAP and its receptors on cumulus cells: effects on oocyte maturation.

Mol Cell Endocrinol 2013 Aug 16;375(1-2):79-88. Epub 2013 May 16.

Department of Anatomy, Histology, Forensic Medicine and Orthopedic, Section of Histology, Sapienza University of Rome, Rome, Italy.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1-R (PACAP type 1 receptor) are transiently expressed in granulosa cells (GCs) of mouse preovulatory follicles and affect several parameters associated with the ovulatory process. We investigated the expression of PACAP and its receptors in cumulus cells (CCs) after the LH surge and their role on cumulus expansion/apoptosis and oocyte maturation. PACAP and PAC1-R expression increased in CCs isolated at different times after treatment with human chorionic gonadotropin (hCG). Moreover, PACAP was able to reverse the inhibition of oocyte meiotic maturation caused by hypoxantine in cumulus cell-oocyte complexes (COCs) and efficiently promoted male pronuclear formation after fertilisation. PACAP was also able to induce cumulus expansion and prevent CC apoptosis. Our results demonstrated the induction of PACAP and its receptors in CCs by LH and EGF, suggesting that PACAP may play a significant role in the complex interactions of gonadotropin and growth factors during ovulation and fertilisation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mce.2013.05.006DOI Listing
August 2013

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1071/RD13010DOI Listing
December 2014

Follicular fluid hormonal profile and cumulus cell gene expression in controlled ovarian hyperstimulation with recombinant FSH: effects of recombinant LH administration.

J Assist Reprod Genet 2012 Dec 28;29(12):1381-91. Epub 2012 Nov 28.

Department of Anatomy, Histology, Forensic Medicine and Orthopedic, Section of Histology, Sapienza University of Rome, Rome, Italy.

Purpose: Down-regulation with gonadodropin-releasing agonist (GnRH-a) protocol during IVF stimulation leads to a severe endogenous LH suppression, which may affect the follicular development. The aim of the study was to evaluate the effects of recombinant LH (r-LH) administration, during late follicular development stages, in recombinant FSH (r-FSH) stimulated cycles on follicular fluid (FF) parameters and on cumulus cell quality.

Methods: Twenty patients undergoing IVF were stimulated in a long GnRH agonist protocol with r-FSH alone or with r-LH supplementation when the leading follicle reached diameter of 14 mm. FF was collected at the time of oocyte retrieval from 32 follicles ≥ 18 mm. Serum FSH, LH, estradiol (E(2)), and progesterone (P(4)) were evaluated on the day of hCG administration. Intra-follicular E(2), P(4), AMH and TGF-β were assayed. Total RNA from 18 individual cumuli was isolated for gene expression analyses.

Results: R-LH increased FF P(4) levels. FF TGF-β levels and PTGS2 and HAS2 expression in cumulus cells (CCs) positively correlated with increased P(4) levels observed in FFs, while a negative correlation was found between P(4) and AMH levels.

Conclusions: FF positive correlation between P(4) and TGF-β levels and CC expression of PTGS2 and HAS2 suggest an association with a better follicle quality. In addition, our data suggest that late follicular phase r-LH supplementation leads to a more advanced stage of follicular maturation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10815-012-9893-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528868PMC
December 2012

Hepatocyte growth factor is a mouse fetal Leydig cell terminal differentiation factor.

Biol Reprod 2012 Jun 21;87(6):146. Epub 2012 Dec 21.

Department of Experimental Medicine, Histology and Embryology Laboratory, School of Medicine, Second University of Naples, Naples, Italy.

The hepatocyte growth factor (HGF) is a pleiotropic cytokine and a well-known regulator of mouse embryonic organogenesis. In previous papers, we have shown the expression pattern of HGF and its receptor, C-MET, during the different stages of testis prenatal development. We demonstrated that C-MET is expressed in fetal Leydig cells (FLCs) and that HGF stimulates testosterone secretion in organ culture of late fetal testes. In the present study, we analyzed the proliferation rate, apoptotic index, and differentiation of FLCs in testicular organ culture of 17.5 days postcoitum (17.5 dpc) embryos to clarify the physiological role of HGF in late testis organogenesis. Based on our data, we conclude the following: 1) HGF acts as an antiapoptotic factor that is able to reduce the number of apoptotic FLCs and testicular caspase-3 active fragment; 2) HGF does not affect FLC proliferation; 3) HGF significantly increases expression of insulin-like 3 (INSL3), a marker of Leydig cell terminal differentiation, without affecting 3beta-hydroxysteroid dehydrogenase (3betaHSD) expression; 4) HGF significantly decreases the expression of nestin, a marker of Leydig cell progenitors; and 5) HGF significantly increases the number of fully developed FLCs. Taken together, these observations demonstrate that HGF is able to act in vitro as a survival and differentiation factor in FLC population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1095/biolreprod.112.104638DOI Listing
June 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.taap.2012.02.005DOI Listing
April 2012

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/tox.20711DOI Listing
April 2013

Inhibitory effect of pituitary adenylate cyclase activating polypeptide on the initial stages of rat follicle development.

Mol Cell Endocrinol 2010 May 6;320(1-2):34-44. Epub 2010 Feb 6.

Department of Histology and Medical Embryology, La Sapienza University of Rome, Via A. Scarpa, 16, Rome 00161, Italy.

Pituitary adenylate cyclase activating polypeptide (PACAP) is transiently expressed in preovulatory follicles of different species and positively affects parameters correlated with the ovulatory process. It has also been shown to be expressed in the interstitial tissue and in interstitial glandular cells in the proximity of primordial and preantral follicles. The aim of the present study was to investigate whether PACAP influences the recruitment of primordial follicles and the growth and differentiation of preantral follicles. Rat ovaries from 2-day-old animals were cultured for 5 days in the presence of PACAP. This treatment significantly inhibited the primordial to primary follicle transition. PACAP inhibited granulosa cell proliferation without affecting cell viability. PACAP also inhibited the growth of isolated preantral follicles cultured under basal conditions or in the presence of follicle-stimulating hormone (FSH). These results suggest that PACAP is significantly involved in the cyclic recruitment of primordial follicles and in the FSH-dependent growth of preantral follicles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mce.2010.01.040DOI Listing
May 2010

XIV workshop on the development and function of reproductive organs.

Int J Dev Biol 2009 ;53(7):883-9

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

The XIV Workshop on the Development and Function of Reproductive Organs was held in Rome, from 14-17 September, 2008, at the Congress Centre of the University of Tor Vergata of Villa Mondragone (Rome, Italy). The Workshop was conceived to be a survey of the events from the formation of the gamete precursors, the primordial germ cells, to the development of the preimplantation embryo through female and male gametogenesis in the mouse experimental model. It was divided into six topics including classic and highly-debated current subjects in the field of the biology of reproduction: mammalian primordial germ cells, the formation of the gonads, stem cells and germ cells, gametogenesis from birth to adult (male), gametogenesis from birth to adult (female), and finally fertilization and preimplantation embryo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1387/ijdb.082816mfDOI Listing
October 2009

Characterization, expression, and functional activity of pituitary adenylate cyclase-activating polypeptide and its receptors in human granulosa-luteal cells.

J Clin Endocrinol Metab 2008 Dec 9;93(12):4924-32. Epub 2008 Sep 9.

Department of Histology and Medical Embryology, "La Sapienza" University of Rome, Via A. Scarpa 14, 00161 Rome, Italy.

Context: Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are found in the ovary of mammalian species, although nothing is known about the possible role of PACAP and VIP in the human ovary.

Objective: We investigated the expression of PACAP and PACAP/VIP receptors in human granulosa-luteal (GL) cells obtained from consenting in vitro fertilization patients attending a private fertility clinic and assessed a possible antiapoptotic effect of these molecules.

Main Outcome Measures: We measured the expression of PACAP and PACAP/VIP receptor mRNAs in GL cells in response to FSH or LH, as well as the effects of PACAP and VIP on apoptosis. We also evaluated the levels of procaspase-3 in GL cells cultured in the absence of serum.

Results: After 7 d in culture, GL cells displayed increased responsiveness to FSH and LH (100 ng/ml). FSH and LH promoted PACAP expression, LH doing so in a time-dependent fashion. VIP receptor (VPAC1-R and VPAC2-R) mRNAs were also induced by gonadotropin stimulation. Although PACAP receptor (PAC1-R) mRNA was barely detectable, Western blot analysis revealed its presence. The apoptotic effect of serum withdrawal from the culture environment was reverted by both PACAP and VIP. Both peptides showed the ability to reverse a decrease in procaspase-3 levels induced by culture in the absence of serum.

Conclusions: PACAP and VIP appear to play a role in maintenance of follicle viability as a consequence of the antiapoptotic effect. Further studies are warranted to evaluate the respective roles of PACAP and VIP in ovarian physiology and to identify their mechanism of action.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/jc.2007-2621DOI Listing
December 2008

Expression localisation and functional activity of pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors in mouse ovary.

Reproduction 2007 Aug;134(2):281-92

Department of Histology and Medical Embriology, La Sapienza University of Rome, Via A. Scarpa, 14, Rome 00161, Italy.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) positively affect several parameters correlated with the ovulatory process. PACAP is transiently expressed in rat preovulatory follicles, while VIP is present in nerve fibres at all stages of development. These two peptides act by interacting with three types of receptors: PACAP type I receptor (PAC1-R), which binds with higher affinity to PACAP, and two VIP receptors (VPAC1-R and VPAC2-R), which bind to PACAP and VIP with equal affinity. The aim of the present study was to characterise the PACAP/VIP/receptor system in the mouse ovary. Results obtained by RT-PCR, immunohistochemistry and in situ hybridisation showed that PACAP was transiently expressed in granulosa cells of preovulatory follicles after human chorionic gonadotrophin (hCG) stimulation, while VIP mRNA was never observed. All the receptors were present in 22-day-old untreated mice. In preovulatory follicles, PAC1-R was expressed both in granulosa cells and in residual ovarian tissue but was stimulated by hCG mainly in granulosa cells; VPAC2-R was present in both the cell compartments and was only mildly stimulated; VPAC1-R was present mainly in the residual ovarian tissue and was downregulated by hCG. PACAP and VIP were equipotent in inhibiting apoptosis in granulosa cells, confirming the presence of functional PACAP/VIP receptors. The contemporary induction by hCG of PACAP and PAC1-R in granulosa cells of preovulatory follicles suggests that, also in mouse ovary, PACAP may play a significant role around the time of ovulation. Moreover, the presence of PACAP/VIP receptors in the untreated ovary suggests a possible role for PACAP and VIP during follicle development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/REP-07-0051DOI Listing
August 2007

Characterization and expression of different pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide receptors in rat ovarian follicles.

J Endocrinol 2006 Oct;191(1):287-99

Department of Histology and Medical Embryology, University of Rome La Sapienza, Via A. Scarpa, 14, 00161 Rome, Italy.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide transiently expressed in preovulatory follicles. PACAP acts by interacting with three types of PACAP receptors. PACAP type I receptor (PAC(1)-R), which binds specifically to both PACAPs and vasoactive intestinal polypeptide (VIP), although with lower affinity, and two VIP receptors, VPAC(1)-R and VPAC(2)-R, which bind to PACAP and VIP with equal affinity. In the present study, we showed the expression of all three receptors in whole ovaries obtained from juvenile and gonadotropin-treated immature rats. A more detailed analysis on cells from preovulatory follicles showed that PAC(1)-R and VPAC(2)-R were expressed in granulosa cells, whereas only VIP receptors were expressed in theca/interstitial (TI) cells and fully grown oocytes presented only PAC(1)-R. The distribution of the VIP receptors was confirmed by immunofluorescence. HCG treatment induced stimulation of PAC(1)-R in granulosa cells and VPAC(2)-R in TI cells. The presence of functional PACAP/VIP receptors was also supported by metabolic studies. We further evaluated the presence of PACAP and VIP receptors by testing the effect of these peptides on apoptosis in granulosa cells cultured, isolated or in whole follicles. Treatment of follicles with PACAP and VIP dose-dependently inhibited apoptosis, while only PACAP significantly inhibited isolated granulosa cells. These results demonstrate a different expression of PACAP/VIP receptors in the various follicle compartments and suggest a possible role for PACAP and VIP on granulosa and TI cells, both during follicle development and ovulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1677/joe.1.06470DOI Listing
October 2006

Urokinase plasminogen activator and TGF-beta production in immunosuppressed patients with and without P. Jiroveci infection.

Microb Pathog 2006 Jul 15;41(1):1-9. Epub 2006 May 15.

Department of Clinical Medicine, University La Sapienza, Rome, Italy.

Macrophages play a pivotal role in a host's defence against pulmonary infections. Macrophage functions are impaired in immunosuppressed (IS) patients, regardless of whether they are HIV-positive (HIV+) or -negative (HIV-). Several studies have indicated that urokinase plasminogen activator (uPA) and transforming growth factor beta (TGF-beta) are important factors in a host's defence against pulmonary pathogens. We measured uPA and TGF-beta activity in unstimulated peripheral blood monocytes (PBM) of both HIV-infected and non-infected IS patients with or without Pneumocystis jiroveci (formerly carinii) pneumonia (PCP). As previously found in alveolar macrophages (AMs), the majority of uPA activity was found in cell lysates. The highest values of uPA activity were found in control subjects. All the patients displayed a decreased production of uPA, irrespective of HIV infection. Similarly, active TGF-beta was higher in control subjects than in HIV+ and IS patients. The presence of P. jiroveci infection further lowered uPA and TGF-beta activity. Decreased TGF-beta activation might be a consequence of lower uPA production, which may, in turn, influence virus replication, since it has been demonstrated that TGF-beta can suppress human HIV expression in monocytes/macrophages. Further studies are warranted to elucidate whether the decrease in uPA and TGF-beta activity impairs a host's defence against P. jiroveci infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.micpath.2006.03.003DOI Listing
July 2006

Effects of insulin-like growth factor I and II on prostaglandin synthesis and plasminogen activator activity in human umbilical vein endothelial cells.

J Clin Endocrinol Metab 2005 Jan 26;90(1):372-8. Epub 2004 Oct 26.

Cattedra di Fisiopatologia della Riproduzione Umana, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, Italy.

IGFs seem to contribute to the endothelial dysfunction observed in some vascular diseases. Because locally increased IGFs levels were detected in the preeclamptic fetoplacental unit, we hypothesized their involvement in the dysregulation of fibrinolysis and vascular tone typically observed in the fetoplacental compartment in this pregnancy disease. Therefore, in human umbilical vein endothelial cells (HUVECs), the potential effect of IGFs on the synthesis of plasminogen activators (PAs), PA inibitor-1 (PAI-1), and vasodilator and vasoconstrictor prostaglandins (PGs) was investigated. Moreover, in HUVECs treated with IGFs, the expression of cyclooxygenase (COX)-2, the rate-limiting enzyme in PG synthesis, was evaluated.HUVECs were treated for 24 h with IGFs (1-100 ng/ml) or IL-1beta (0.1 ng/ml). PA, PAI-1, and COX-2 mRNA was determined by RT-PCR and PG release and PA activity by RIA and colorimetric assay, respectively.We demonstrated an inhibition of urokinase-type PA activity and a 50% reduction of urokinase-type PA mRNA in HUVECs treated with IGFs. No effect was seen on PAI-1. Finally, both IGFs significantly decreased all PGs tested and COX-2 mRNA, whereas, as expected, IL-1beta had an opposite effect. In conclusion, our results suggest for IGFs a potential involvement in the endothelial dysfunction observed in preeclamptic fetoplacental unit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/jc.2004-1022DOI Listing
January 2005

Granulosa cell-oocyte interactions.

Eur J Obstet Gynecol Reprod Biol 2004 Jul;115 Suppl 1:S19-22

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

Throughout oogenesis the oocyte and follicle cells establish an intricate system of mutual interactions that ultimately lead to the acquisition of their respective competences. Paracrine factors released by both cell types are believed to stimulate formation of the primordial follicle and support the initial phases of follicle growth. At the same time, these processes are also dependent on gap junction communication between the germinal and somatic compartment. At later stages of follicle development, activities released by the oocyte induce the adjacent granulosa cells to express a specialized phenotype. In their turn, these cells crucially regulate the ability of the oocyte to progress through the meiotic process and acquire full developmental potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejogrb.2004.01.010DOI Listing
July 2004

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2003-1004DOI Listing
April 2004

Pituitary adenylate cyclase-activating polypeptide modulates plasminogen activator expression in rat granulosa cell.

Biol Reprod 2002 Mar;66(3):830-5

Department of Obstetrics and Gynecology, Università Cattolica del Sacro Cuore, Rome, Italy.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide isolated from ovine hypothalamus. It has been demonstrated to be transiently expressed in preovulatory follicles and to positively affect several parameters correlated with the ovulatory process. The aim of the present study was to investigate whether PACAP influences the plasminogen/plasmin system in rat ovary. Plasminogen activators (PAs) are serine proteases, modulated by gonadotropins and several peptides in preovulatory follicles, that appear to be involved in ovulation. Granulosa cells obtained from immature eCG-treated rats were cultured for 24 h in the presence of increasing concentrations of PACAP and vasoactive intestinal peptide (VIP). A significant, dose-dependent increase in tissue-type PA (tPA) activity and decrease in urokinase-type (uPA) PA activity were observed in PACAP-treated cells. These effects were exerted at the mRNA level. The use of cycloheximide, a protein synthesis inhibitor, suggested that PACAP requires an intermediary protein to decrease uPA-mRNA, but not to induce tPA-mRNA. However, no significant modulation of PAs was observed in the presence of VIP. When granulosa cells were stimulated within the intact follicle (i.e., maintaining the three-dimensional structure and in the presence of the theca cell layers), both PACAP and VIP dose-dependently stimulated tPA. These data suggest that, in addition to the PACAP type I receptor present on granulosa cells, different subtypes of PACAP receptors are present in the different ovarian compartments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1095/biolreprod66.3.830DOI Listing
March 2002