Gianna Rossi

The fungicide mancozeb induces toxic effects on mammalian granulosa cells.

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.

The Effects of the Endocrine Disruptors Dithiocarbamates on the Mammalian Ovary with Particular Regard to Mancozeb

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.

Mammalian oocyte growth in vitro is stimulated by soluble factor(s) produced by preantral granulosa cells and by Sertoli cells

We have evaluated the possibility that mouse oocyte growth in vitro could be achieved under the influence of soluble compound(s) released by different somatic cell types. For this purpose, zona‐free denuded oocytes from 12‐day‐old mice were cultured on monolayers of NIH‐3T3 fibroblasts, which are able to establish gap junctional communications with them, in the presence or absence of media conditioned by preantral granulosa cells or by Sertoli cells, plated at increasing concentrations from 0.3–1 × 106 ml−1 cells. After 3 days, no increase in vitellus diameter was recorded from fibroblast‐coupled oocytes maintained in culture medium or in the presence of media conditioned by 0.3 × 106 ml−1 Sertoli cells. By contrast, increasing proportions of coupled oocytes grew, provided the continuous presence of media conditioned by 0.5 or 1 × 106 ml−1 Sertoli cells, or by 0.3, 0.5, and 1 × 106 ml−1 preantral granulosa cells. Since the ligand of c‐kit, the growth factor KL, promotes the growth in vitro of oocytes cultured in follicles from 8‐day‐old mice, an antibody against mouse KL was used to evaluate whether in our culture conditions KL might also be responsible for the growth of oocytes from 12‐day‐old mice. No inhibition of growth was evident in oocytes cultured directly on preantral granulosa or Sertoli‐cell monolayers. Furthermore, the growth of fibroblast‐coupled oocytes cultured in media conditioned by preantral granulosa cells was not significantly affected by the presence of this antibody during culture. By contrast, a high percentage of oocytes cultured on fibroblasts in the presence of media conditioned by Sertoli cells showed a significant inhibition of growth and no metabolic cooperativity. It was concluded that, besides KL, other bioactive factor(s) released by either preantral granulosa or Sertoli cells can induce a significant stimulation of mouse oocyte growth in vitro.

Ultrastructure of isolated mouse ovarian follicles cultured in vitro.

BackgroundIn 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.MethodsThe 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.ResultsThe 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).ConclusionsIt 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.

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

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.

Akt expression in mouse oocytes matured in vivo and in vitro

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.

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

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.

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

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.

Endocannabinoid signaling in mammalian ovary

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.

Presence of a 31-kD protein band in human cumulus–corona radiata–conditioned media and pregnancy outcome

OBJECTIVE To investigate relationships between cumulus-oocyte complex (COC) morphology, protein patterns of cumulus-corona (CC) cell-conditioned media, and pregnancy outcome in IVF-ET cycles. DESIGN Retrospective study. SETTING Private university IVF center. PATIENT(S) One hundred twenty infertile women who underwent IVF-ET procedures. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) COC grading, analysis of CC cell morphology and conditioned media, and pregnancy rate (PR). RESULT(S) After IVF and embryo transfer, cultured CC cells were classified as high (HA) or low (LA) on the basis of their adhesive properties. Neither adhesion activity nor fertilization rates and embryo quality were correlated with COC grading. PR in cycles with HA cells was 38%, but 14% of cycles showing LA activity also had positive outcome. To find more meaningful parameters of CC cells useful to predict fertilization and pregnancy, the electrophoretic protein patterns of media conditioned by HA or LA cells were studied. Retrospective analysis showed that all cycles in which replaced embryos were associated with the presence of a 31-kD band in conditioned media failed implantation, whereas 83% of cycles lacking this band resulted in positive, ongoing pregnancy. CONCLUSION(S) Pregnancy prediction cannot rely simply on CC cell morphological analysis. Screening of conditioned media may provide more reliable parameters.