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Malathion-Induced Granulosa Cell Apoptosis in Caprine Antral Follicles: An Ultrastructural and Flow Cytometric Analysis

Published online by Cambridge University Press:  20 November 2014

Jitender K. Bhardwaj  and
Priyanka Saraf
Jitender K. Bhardwaj*
Affiliation:
Reproductive Physiology Laboratory, Department of Zoology, Kurukshetra University, Kurukshetra-136119, Haryana, India
Priyanka Saraf
Affiliation:
Reproductive Physiology Laboratory, Department of Zoology, Kurukshetra University, Kurukshetra-136119, Haryana, India
*
*Corresponding author. jkbkuk@gmail.com
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Abstract

Organophosphate pesticides (OPs) like malathion interfere with normal ovarian function resulting in an increased incidence of atresia and granulosa cell apoptosis that plays a consequential role in the loss of ovarian follicles or follicular atresia. The aim of present study was to assess malathion-induced (100 nM) reproductive stress, ultrastructural damage and changes in apoptosis frequency in ovarian granulosa cells of antral follicles. Transmission electron microscopy (TEM) was employed for ultrastructural characterization, oxidative stress was evaluated using thiobarbituric acid reactive substances (TBARS) assay to measure lipid peroxidation, and apoptosis was quantified via flow cytometry. By TEM, apoptosis was identified by the presence of an indented nuclear membrane with blebbing, pyknotic crescent-shaped fragmented nuclei, increased vacuolization, degenerating mitochondria, and lipid droplets. The results indicate a significant increase in malondialdehyde (MDA) level (nmols/g wet tissue) at a 100 nM dose of malathion i.e. 7.57±0.033*, 8.53±0.12*, and 12.87±0.78** at 4, 6, or 8 h, respectively, as compared with controls (6.07±0.033, p<0.01*, p<0.05**) showing a positive correlation between malathion-induced lipid peroxidation and percentage of granulosa cell apoptosis (r=1; p<0.01). The parallel use of these three methods enabled us to determine the role of malathion in inducing apoptosis as a consequence of cytogenetic damage and oxidative stress generated in granulosa cells of antral follicles.

Keywords

apoptosismalathionoxidative stressultrastructure
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 6 , December 2014 , pp. 1861 - 1868
Copyright
© Microscopy Society of America 2014 

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References

Aktar, M.D., Sengupta, D. & Chowdhury, A. (2009). Impact of pesticides use in agriculture: Their benefits and hazards. Interdiscip Toxicol 2(1), 1–12.CrossRefGoogle ScholarPubMed
Albertini, D.F. & Anderson, E. (1974). The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. J Cell Biol 63, 234–250.CrossRefGoogle ScholarPubMed
Andersen, H.R., Vinggaard, A.M., Rasmussen, T.H., Gjermandsen, I.M. & Bonefeld-Jørgensen, E.C. (2002). Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicol Appl Pharmacol 179, 1–12.CrossRefGoogle ScholarPubMed
Araújo, V.R., Lima-Verde, I.B., Olazia-Name, K.P., Báo, S.N., Campello, C.C., Silva, J.R.V., Rodrigues, A.P.R. & Figueiredo, J.R. (2010). Bone morphogenetic protein-6 (BMP-6) induces atresia in goat primordial follicles cultured in vitro. Pesq Vet Bras 30(9), 770–776.CrossRefGoogle Scholar
Asmathbanu, I. & Kaliwal, B.B. (1997). Temporal effect of methyl parathion on ovarian compensatory hypertrophy, follicular dynamics and estrous cycle in hemi-castrated albino rats. J Basic Clin Physiol Pharmacol 8, 237–254.CrossRefGoogle Scholar
Banerjee, S., Banerjee, S., Saraswat, G., Bandyopadhyay, S.A. & Kabir, S.N. (2014). Female reproductive aging is master-planned at the level of ovary. PLoS One 9(5), e96210. doi:10.1371/journal.pone.0096210CrossRefGoogle ScholarPubMed
Beckman, K.B. & Ames, B.N. (1998). The free radical theory of aging matures. Physiol Rev 78, 547–581.CrossRefGoogle ScholarPubMed
Behrman, H.R. & Preston, S.L. (1988). Luteolytic actions of peroxide in rat ovarian cells. Endocrinology 124, 2895–2900.CrossRefGoogle Scholar
Blasiak, J., Jaloszynski, P., Trzeciak, A. & Szyfter, K. (1999). In vitro studies on the genotoxicity of the organophosphorus insecticide malathion and its two analogues. Mutat Res 445(2), 275–283.CrossRefGoogle ScholarPubMed
Bonilla, E., Hernandez, F., Cortes, L., Mendoza, M., Mejia, J., Carrillo, E., Casas, E. & Betancourt, M. (2007). Effects of the insecticides malathion and diazinon on the early oogenesis in mice in vitro . Environ Toxicol 23, 240–245.CrossRefGoogle Scholar
Buege, J.A. & Aust, S.D. (1978). Microsomal lipid peroxidation. Meth Enzymol 51, 302–310.CrossRefGoogle Scholar
Carbone, M.C., Tatone, C., Delle, M.S., Marci, R., Caserta, D., Colonna, R. & Amicarelli, F. (2003). Antioxidant enzymatic defenses in human follicular fluid: Characterization and age-dependent changes. Mol Hum Reprod 9, 639–643.CrossRefGoogle ScholarPubMed
Centurione, L., Giampietro, F., Sancilio, S., Piccirilli, M., Artese, L., Tiboni, G.M. & Di-Pietro, R.D. (2010). Morphometric and ultrastructural analysis of human granulosa cells after gonadotrophin-releasing hormone agonist or antagonist. Reprod BioMed Online 20, 625–633.CrossRefGoogle ScholarPubMed
Chitnis, S.S., Navlakhe, R.M., Shinde, G.C., Barve, S.J., D’souza, S., Mahale, S.D. & Nandedkar, T.D. (2008). Granulosa cell apoptosis induced by a novel fsh binding inhibitory peptide from human ovarian follicular fluid. J Histochem Cytochem 56(11), 961–968.CrossRefGoogle ScholarPubMed
Devine, J., Payne, C.M., Mccuskey, M.K. & Hoyer, P.B. (2000). Ultrastructural evaluation of oocytes during atresia in rat ovarian follicles. Biol Reprod 63, 1245–1252.CrossRefGoogle ScholarPubMed
Devine, P.J., Perreault, S.D. & Luderer, U. (2012). Roles of reactive oxygen species and antioxidants in ovarian toxicity. Biol Reprod 86(2), 1–10.CrossRefGoogle ScholarPubMed
Escobar, M.L., Echeverría, O.M., Casasa, A.S., García, G., Aguilar, S.J. & Vázquez-Nin, G.H. (2013). Involvement of pro-apoptotic and pro-autophagic proteins in granulosa cell death. Cell Biol 1(1), 9–17.Google Scholar
Eskenazi, B., Marks, A.R., Bradman, A., Harley, K., Barr, D.B., Johnson, C., Morga, N. & Jewel, N.P. (2007). Organophosphate pesticide exposure and neurodevelopment in young Mexican-American children. Environ Health Perspect 115, 792–798.CrossRefGoogle ScholarPubMed
Gavrieli, Y., Sherman, Y. & Ben-Sasson, S.A. (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119, 493–501.CrossRefGoogle ScholarPubMed
Guney, M., Dermirin, H., Oral, B., Ozguner, M., Bayhan, G. & Altuntas, I. (2007). Ovarian toxicity in rats caused by methidathion and ameliorating effects of vitamins E and C. Hum Exp Toxicol 26, 491–498.CrossRefGoogle ScholarPubMed
Hay, M.F., Cran, D.G. & Moor, R.M. (1976). Structural changes occurring during atresia in sheep ovarian follicles. Cell Tiss Res 169, 515–529.CrossRefGoogle ScholarPubMed
Hertig, A. & Adams, E. (1967). Studies on human oocyte and its follicle, ultrastructure and its cytochemical observation on the preovulatory follicles. J Cell Biol 34, 647–675.CrossRefGoogle Scholar
Hyttel, P., Westergaard, L. & Byskov, A.G. (1986). Ultrastructure of human cumulus–oocyte complexes from healthy and atretic follicles. Hum Reprod 1, 153–157.CrossRefGoogle ScholarPubMed
Janero, D. (1990). Malondialdehyde and thiobarbituric acid reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 9, 515–540.CrossRefGoogle ScholarPubMed
Kamanyire, R. & Karallidde, L. (2004). Organophosphate toxicity and occupational exposure. Occup Med 54, 65–76.CrossRefGoogle ScholarPubMed
Keren-Tal, J., Suh, B.S., Dantes, A., Lindner, S., Oren, M. & Amsterdam, A. (1995). Involvement of p53 expression in cAMP-mediated apoptosis in immortalized granulosa cells. Exp Cell Res 218, 283–295.CrossRefGoogle ScholarPubMed
Koc, N.D., Kayhan, F.E., Sesal, C. & Muslu, M.N. (2009). Dose-dependent effects of endosulfan and malathion on adult Wistar albino rat ovaries. Pak J Boil Sci 12(6), 498–503.CrossRefGoogle ScholarPubMed
Lima-Verde, I.B., Matos, M.H.T., Celestino, J.J.H., Rossetto, R., Name, K.P.O., Báo, S.N., Campello, C.C. & Figueiredo, J.R. (2012). Progesterone and follicle stimulating hormone interact and promote goat preantral follicles survival and development in vitro. Pesq Vet Brasil 32(4), 361–367.CrossRefGoogle Scholar
Makrigiannakis, A., Coukos, G., Christofidou-Solomidou, M., Gour, B.J., Radice, G.L., Blaschuk, O. & Coutifaris, C. (1999). N-cadherin-mediated human granulosa cell adhesion prevents apoptosis: A role in follicular atresia and luteolysis? Am J Pathol 154(5), 1391–1406.CrossRefGoogle ScholarPubMed
Merk, F.B., Botticelli, C.R. & Albright, J.T. (1972). An intercellular response to estrogen by granulosa cells in the rat ovary: An electron microscope study. Endocrinology 90, 992–1007.CrossRefGoogle ScholarPubMed
Miller, K.P., Gupta, R.K., Greenfeld, C.R., Babus, J.K. & Flaws, J.A. (2005). Methoxychlor directly affects ovarian antral follicle growth and atresia through Bcl-2- and bax mediated pathways. Toxicol Sci 88(1), 213–221.CrossRefGoogle ScholarPubMed
Okatani, Y., Morioka, N., Wakatsuki, A., Nakano, Y. & Sagara, Y. (1993). Role of the free radical-scavenger system in aromatase activity of the human ovary. Horm Res 39, 22–27.CrossRefGoogle ScholarPubMed
Pangas, S.A. (2007). Growth factors in ovarian development. Semin Reprod Med 25, 225–234.CrossRefGoogle ScholarPubMed
Peluso, J.J., Charlesworth, C., Bolender, D.L. & Steger, R.W. (1980). Ultrastructural alterations associated with the initiation of follicular atresia. Cell Tiss Res 211, 105–115.CrossRefGoogle ScholarPubMed
Peluso, J.J. & Steger, R.W. (1977). Surface ultrastructural changes in granulosa cells of atretic follicles. Biol Reprod 16, 600–604.CrossRefGoogle ScholarPubMed
Ricci, J.E., Muñoz-Pinedo, C., Fitzgerald, P., Bailly-Maitre, B., Perkins, G.A., Yadava, N., Scheffler, I.E., Ellisman, M.H. & Green, D.R. (2004). Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain. Cell 117, 773–786.CrossRefGoogle ScholarPubMed
Rocket, J.C., Narotsky, M.G., Thomson, K.E., Thilladarajah, I., Blystone, C.B., Goetza, A., Rena, H., Besta, D.S., Murrella, R.N., Nicholsa, H.P., Schmida, J.E., Wolfa, D.C. & Dixa, D.J. (2006). Effect of conozole fungicides on reproductive development in female rats. Reprod Toxicol 22, 647–648.CrossRefGoogle Scholar
Samuel, J.B., Stanley, J.A., Princess, R.A., Shanthi, P. & Sebastian, M.S. (2011). Gestational cadmium exposure-induced ovotoxicity delays puberty through oxidative stress and impaired steroid hormone levels. J Med Toxicol 7, 195–204.CrossRefGoogle ScholarPubMed
Santos, H.B., Sato Moro, L.Y., Bazzoli, N. & Rizzo, E. (2008). Relationship among follicular apoptosis, integrin beta1 and collagen type IV during early ovarian regression in the teleost Prochilodus argenteus after induced spawning. Cell Tiss Res 332(1), 159–170.CrossRefGoogle ScholarPubMed
Sawada, M. & Carlson, J.C. (1996). Intracellular regulation of progesterone secretion by the superoxide radical in the rat corpus luteum. Endocrinology 137, 1580–1584.CrossRefGoogle ScholarPubMed
Scandalios, J.G. (2005). Oxidative stress: Molecular perception and transduction of signals triggering antioxidant gene defenses. Braz J Med Biol Res 38, 995–1014.CrossRefGoogle ScholarPubMed
Seth, V., Banerjee, B.D., Ahmed, R.S., Bhattacharya, A. & Pasha, S.T. (2008). Alterations in immunoglobulins and cytokine level in blood of malathion poisoning cases. Indian J Biochem Biophys 45, 209–211.Google Scholar
Sharma, R.K. (2000). Follicular atresia in goat: A review. Ind J Anim Sci, 1035–1046.Google Scholar
Sharma, R.K. & Bhardwaj, J.K. (2009). Ultrastructural characterization of apoptotic granulosa cells in caprine cells. J Microsc 236, 236–242.CrossRefGoogle Scholar
Tilly, J.L., Kowalski, K., Johnson, A.L. & Hsueh, A.J.W. (1991). Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 129, 2799–2801.CrossRefGoogle ScholarPubMed
Venerosi, A., Ricceri, L., Scattoni, M.L. & Calamandrei, G. (2009). Prenatal chlorpyrifos exposure alters motor behavior and ultrasonic vocalization in CD-1 mouse pups. Environ Health 8, 12–23.CrossRefGoogle ScholarPubMed
Wójtowicz, A.K., Gregoraszczuk, E.L., Ptak, A. & Falandysz, J. (2004). Effect of single and repeated in vitro exposure of ovarian follicles to o,p’-DDT and p,p’-DDT and their metabolites. Pol J Pharmacol 56, 465–472.Google Scholar
Yang, M.Y. & Rajamahendran, R. (2000). Involvement of apoptosis in the atresia of non-ovulatory dominant follicle during the bovine estrous cycle. Biol Reprod 62, 1209–1217.CrossRefGoogle Scholar
Yu, Y.S., Sui, H.S., Han, Z.B., Li, W., Luo, M.J. & Tan, J.H. (2004). Apoptosis in granulosa cells during follicular atresia: Relationship with steroids and insulin-like growth factors. Cell Res 14(4), 341–346.CrossRefGoogle ScholarPubMed
Zuelke, K.A., Jones, D.P. & Perreault, S.D. (1997). Glutathione oxidation is associated with altered microtubule function and disrupted fertilization in mature hamster oocytes. Biol Reprod 57, 1413–1419.CrossRefGoogle ScholarPubMed