Effects of the pesticide lindane on granulosa cell ultrastructure

The excessive exposure to pesticides in the Aral Sea area was correlated to the increased reproductive pathologies in those regions. One of the principal chemical employed was the gamma-hexachlorocycloexane herbicide Lindane (L), a persistent organochlorine that may induces alterations in granulosa cell (GCs) survival. However, a comprehensive experimental study on the L-induced dose-effect morphological alterations, has not yet addressed. Therefore, we studied by means of transmission and scanning electron microscopy, the morphological changes of mouse GCs, matured in vitro with increasing concentrations of L. GCs showed several dose-dependent changes, in respect to controls. In particular, we observed significant reduction of GC microvilli and decrease of cytoplasmic processes between adjacent GCs. In addition, peripheral aggregation of chromatin under the nuclear membrane, extensive plasma membrane blebbing, abundant GC remnants and cellular debris were also present. Mitochondria, endoplasmic reticula and Golgi apparatuses did not show significant changes. In conclusion, our results showed a dose-dependent toxicity of L on GCs, associated to morphological signs of apoptosis. Alterations of GCs may be associated to impaired oocyte competence and sterility

Morphological changes of the granulosa cells of mice under the hexachlorocyclohexane (lindane) influence

The excessive exposure to pesticides in the Aral Sea area was correlated to the increased reproductive pathologies in those regions [1]. One of the principal chemical employed was the gamma-hexachlorocycloexane herbicide Lindane (L), a persistent organochlorine that may induces alterations in granulosa cell (GCs) survival [2, 3]. However, a comprehensive experimental study on the L-induced dose-effect morphological alterations, has not yet addressed. Therefore, we studied by means of transmission and scanning electron microscopy, the morphological changes of mouse GCs, matured in vitro with increasing concentrations of L [4-6]. GCs showed several dose-dependent changes, in respect to controls. In particular, we observed significant reduction of GC microvilli and decrease of cytoplasmic processes between adjacent GCs. In addition, peripheral aggregation of chromatin under the nuclear membrane, extensive plasma membrane blebbing, abundant GC remnants and cellular debris were also present. Mitochondria, endoplasmic reticula and Golgi apparatuses did not show significant changes. In conclusion, our results showed a dose-dependent toxicity of L on GCs, associated to morphological signs of apoptosis. Alterations of GCs may be associated to impaired oocyte competence and sterility [7]. References [1] Ataniyazova O et al. (2001) Continuing progressive deterioration of the environment in the Aral Sea Region: disastrous effects on mother and child health. Acta Paediatr 90:589-91. [2] Li R, Mather JP. Lindane, an inhibitor of gap junction formation, abolishes oocyte directed follicle organizing activity in vitro. Endocrinology 138:4477-80. [3] Picard A et al. (2003) Effect of organochlorine pesticides on maturation of starfish and mouse oocytes. Toxicol Sci 73:141-8. [4] Rossi G et al (2006) Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes. Reprod Toxicol 22:51-5. [5] Palmerini MG et al (2014) Ultrastructure of Immature and Mature Human Oocytes after Cryotop Vitrification. J Reprod Dev 60:411-20. [6] Martelli A et al. (2009). Blood vessel remodeling in pig ovarian follicles during the periovulatory period: an immunohistochemistry and SEM-corrosion casting study. Reprod Biol Endocrinol 7:72. [7] Kidder GM, Vanderhyden BC (2010). Bidirectional communication between oocytes and follicle cells: ensuring oocyte developmental competence. Can J Physiol Pharmacol 88:399-413

Effects of environmental pollutants on cultured mouse oocytes and granulosa cells

The drying of the Aral Sea for irrigation, followed by an excessive pesticide exposure, is one of the greatest man-made environmental disaster. High levels of reproductive pathologies have been observed in this region for more than 20 years [1,2]. The responsible include Hexachlorocyclohexanes, as the herbicide Lindane, and Dithiocarbamates, as the fungicide Mancozeb. We demonstrated the toxic effect of Mancozeb on mouse oocytes [3,4] characterized by alterations in spindle morphology, a reduction in the formation of male/female pronuclei and a reduced ability to complete meiotic maturation and fertilization. Lindane altered the formation of the first meiotic spindle and polar body extrusion on mouse oocytes in vitro [5]. This was probably due to gap junction inhibition between oocyte and surrounding granulosa cells (GCs) [6]. To understand the ultrastructural morphological alterations on GCs after pesticide exposure, increasing dose of Lindane and Mancozeb were tested on GCs collected from prepubertal CD1 mice ovaries and matured in vitro [3] and studied by transmission and scanning electron microscopies (TEM and SEM) [7,8]. Results indicated dose-dependent detrimental effects on GCs subjected to Lindane or Mancozed exposure, as per the presence of abundant presence of cell debris, the lost of cell contacts, the decrease of microvilli and cytoplasmic processes. In addition, peripheral aggregation of chromatin under the nuclear membrane, extensive plasma membrane blebbing, abundant GC remnants were also present, but more evident after Lindane exposure. In conclusion, our data showed a different dose-dependent toxicity for Lindane and Mancozeb on GCs, associated to morphological signs of cell death. Since alterations of GCs may be associated to impaired oocyte competence and sterility [9], these results may be of interest in the studies addressed to the preservation of fertility, both in animals and humans

Ultrastructure of mouse granulosa cells exposed in vitro to the fungicide Mancozeb

Mancozeb is an ethylene bis-dithiocarbamate widely used as fungicide, also due to a low reported toxicity in mammals. However, reproductive toxicity has been demonstrated in vivo and in vitro in mouse oocytes, by the alteration of spindle morphology [1] and impairment of fertilizability [2]. Mancozeb exerted on mouse GCs cultured in vitro a premalignant-like status, indicated by reduced p53 expression [3] and a mild oxidative stress [4]. However, presence and extent of ultrastructural alterations induced in vitro by Mancozeb on GCs were not yet studied. To this aim, Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) were applied on mouse GCs cultured with increasing concentration of Mancozeb. GCs were obtained by puncturing antral follicles of PMSG-treated prepubertal CD1 female mice and cultured in vitro in DMEM+5%FBS+pen/strep without (control) or with increasing concentration of Mancozeb (0.001-to-1 µg/ml) for 48hrs, at 37°C and 5%CO2. At the end of the culture period, cells were washed in PBS, fixed in 2.5% glutaraldehyde/ PBS and stored at 4°C until processing. GCs were, then, subjected to standard preparative for TEM [5] and SEM [6] observation. Results showed a dose-dependent toxicity of Mancozeb on mouse GCs. Ultrastructural data showed intercellular communication retraction, irregular nuclear membrane and chromatin marginalization at lower concentrations; chromatin condensation, membrane blebbing and cytoplasmic vacuolization at higher concentrations. In conclusion, Mancozeb showed a dose-dependent harmful effect on granulosa cells in vitro, probably due to the toxic breakdown product ethylenethiourea. TEM and SEM were again confirmed to be a valuable tool to study ultrastructural alterations after toxicants exposure

The pesticide Lindane induces dose-dependent damage to granulosa cells in an in vitro culture

Lindane, which is one of the most persistent organochlorine pesticide contaminating the Aral Sea region, is associated with numerous pathologies of the female reproductive system, including infertility, due to its gap junction blocker activity. By using an in vitro model of reproductive toxicity consisting of mouse parietal granulosa cells (GCs) exposed to increasing concentrations of Lindane ranging from 1 to 100μM (L1; L10; L100), we aimed to ascertain the Lindane toxicity by evaluating the ultrastructure and expression of the cell death protein p53. GCs exposed to L1 showed an early sign of degeneration as chromatin marginalization and initial reduction of cell-to-cell contacts. Such effects increased at L10 with nuclear membrane invagination, cytoplasmic blebbing, reduction of microvilli and intercellular connections. L100 induced evident cellular damages with an extensive presence of vacuoles, cytoplasmic fragments, nuclear membrane vesiculation and abundant cellular debris. A dose-dependent increase of p53 expression was evident in the L1 and L10 groups but not in L100. These data provide evidence for a dose-dependent reproductive toxicity of the gap junction blocker Lindane, as seen in mouse GCs cultured in vitro by ultrastructural damage compatible with apoptosis. Since gap junctions may play a critical role in FSH-stimulated progesterone production, the ultrastructural damage here evidenced could explain the increase in the prevalence of reproductive pathologies and infertility in exposed women. Finally, this study provided a useful and repeatable model of reproductive toxicity in vitro, which is applicable to evaluate the detrimental effects of toxicants or the reversing effect of protective substances

The cooperation agreement beetwen the university of l'Aquila, Italy and the Marat Ospanov west-Kazakistan stare medical university, the republic of Kazakistan: a successful story fron academy to bench

During the spring-summer of 2014, the first exchange program foreseen the research mobility from Kazakhstan to Italy. An academic staff member and a PhD student of the Marat Ospanov WestKazakhstan State Medical University spent three months of research internship to the Laboratories of Reproductive Biotechnology, University of L’Aquila. The research topic focused on one of the most dramatic environmental problems, the desiccation of the Aral Sea, which is having serious effects on the human health due to the consequent abuse of pesticides for agricultural purposes. The project, activated in the framework of the Cooperation Agreement, was finalized to study the female reproductive toxicity, with a morphological approach, of one of the most persistent pesticide found in the Aral Sea area, the γ-exachloroexane or Lindane. Thank to the use of Scanning Electron Microscopy and Transmission Electron Microscopy of the Centre for Electron Microscopies, University of L’Aquila and the Laboratory of Electron Microscopy “Pietro Motta”, La Sapienza University of Rome, it have been producing evidences of the dose-dependant harmful effects against granulosa cells and oocytes in mammals. Part of results has been used for an undergraduate thesis and a PhD dissertation. The study is still ongoing but preliminary results have been diffused to national (Italian) and international Congresses as oral communication and poster presentations. Manuscripts to be submitted in peer reviewed journals are in preparation