Induction of apoptotic cell death via accumulation of autophagosomes in rat granulosa cells

This study evaluated the effect of autophagosome accumulation on apoptotic cell death in granulosa cells from developing follicles. Our results indicate that the accumulation of autophagosomes induces apoptotic cell death of granulosa cells through decreased bcl-2 expression and subsequent caspase activation. This study evaluated the effect of autophagosome accumulation on apoptotic cell death in granulosa cells from developing follicles. Our results indicate that the accumulation of autophagosomes induces apoptotic cell death of granulosa cells through decreased bcl-2 expression and subsequent caspase activation. In the mammalian ovary, only a small fraction of follicles mature fully and are involved in ovulation; most follicles become atretic and die. Although follicular atresia occurs repeatedly during the ovarian cycle, the precise mechanism underlying the massive cell death has not been fully elucidated. Recent studies have suggested that follicular atresia is associated with granulosa cell death by apoptosis, or type 1 programmed cell death (PCD) (1Boone D.L. Yan W. Tsang B.K. Identification of a deoxyribonuclease I-like endonuclease in rat granulosa and luteal cell nuclei.Biol Reprod. 1995; 53: 1057-1065Crossref PubMed Scopus (39) Google Scholar, 2Boone D.L. Tsang B.K. Caspase-3 in the rat ovary: localization and possible role in follicular atresia and luteal regression.Biol Reprod. 1998; 58: 1533-1539Crossref PubMed Scopus (167) Google Scholar, 3Flaws J.A. Kugu K. Trbovich A.M. DeSanti A. Tilly K.I. Hirshfield A.N. et al.Interleukin-1 beta-converting enzyme-related proteases (IRPs) and mammalian cell death: dissociation of IRP-induced oligonucleosomal endonuclease activity from morphological apoptosis in granulosa cells of the ovarian follicle.Endocrinology. 1995; 136: 5042-5053Crossref PubMed Google Scholar, 4Hsu S.Y. Lai R.J. Finegold M. Hsueh A.J. 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According to previous studies, apoptosis and autophagy are viewed as clearly distinct subroutines of cellular demise according to morphologic criteria (9Gozuacik D. Kimchi A. Autophagy as a cell death and tumor suppressor mechanism.Oncogene. 2004; 23: 2891-2906Crossref PubMed Scopus (1275) Google Scholar, 10Lockshin R.A. Zakeri Z. Apoptosis, autophagy, and more.Int J Biochem Cell Biol. 2004; 36: 2405-2419Crossref PubMed Scopus (613) Google Scholar, 11Jia L. Dourmashkin R.R. Allen P.D. Gray A.B. Newland A.C. Kelsey S.M. Inhibition of autophagy abrogates tumour necrosis factor alpha induced apoptosis in human T-lymphoblastic leukaemic cells.Br J Haematol. 1997; 98: 673-685Crossref PubMed Scopus (211) Google Scholar). Apoptosis is characterized morphologically by nuclear condensation (pyknosis) and fragmentation (karyorhexis), without major ultrastructural changes of cytoplasmic organelles (12Xue L. Fletcher G.C. Tolkovsky A.M. Autophagy is activated by apoptotic signalling in sympathetic neurons: an alternative mechanism of death execution.Mol Cell Neurosci. 1999; 14: 180-198Crossref PubMed Scopus (391) Google Scholar). Autophagy is defined by the cytoplasmic accumulation of autophagosomes, double-membrane bound vacuoles, the formation of which initiates the autophagic process. The autophagosomes then undergo maturation and fusion with lysosomes for degradation (13Bauvy C. Gane P. Arico S. Codogno P. Ogier-Denis E. Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29.Exp Cell Res. 2001; 268: 139-149Crossref PubMed Scopus (133) Google Scholar, 14Longo L. Platini F. Scardino A. Alabiso O. Vasapollo G. Tessitore L. Autophagy inhibition enhances anthocyanin-induced apoptosis in hepatocellular carcinoma.Mol Cancer Ther. 2008; 7: 2476-2485Crossref PubMed Scopus (115) Google Scholar). In spite of these differences, apoptosis and autophagy are considered to represent parts of a continuum of cell death mechanisms because the induction of apoptotic cell death is regulated by the autophagic process. Apoptotic cell death is induced by inhibiting the accumulation of autophagosomes in various carcinoma cells (15Xue L. Fletcher G.C. Tolkovsky A.M. Mitochondria are selectively eliminated from eukaryotic cells after blockade of caspases during apoptosis.Curr Biol. 2001; 11: 361-365Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 16Kanzawa T. Kondo Y. Ito H. Kondo S. Germano I. Induction of autophagic cell death in malignant glioma cells by arsenic trioxide.Cancer Res. 2003; 63: 2103-2108PubMed Google Scholar), which suggests that the autophagic process prevents apoptotic cell death. However, recent studies have demonstrated that the autophagic process can also induce apoptotic cell death. Studies in human colon adenocarcinoma (17Rao I.M. Mills T.M. Anderson E. Mahesh V.B. Heterogeneity in granulosa cells of developing rat follicles.Anat Rec. 1991; 229: 177-185Crossref PubMed Google Scholar) and HeLa cells (18Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. et al.LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5554) Google Scholar, 19Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation.J Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1144) Google Scholar) have shown that apoptotic cell death is promoted by the accumulation of autophagosomes in the cytoplasm. These findings suggest that the effect of the autophagic process on apoptotic cell death varies depending on cell type, although in all cases it appears that the accumulation of autophagosomes is a crucial process for the regulation of apoptotic cell death. Furthermore, the effect of the autophagic process on apoptotic cell death of granulosa cells in particular has not been elucidated to date, although a recent study has suggested that autophagy is closely related to induction of apoptosis in granulosa cells (8Choi J.Y. Jo M.W. Lee E.Y. Yoon B.K. Choi D.S. The role of autophagy in follicular development and atresia in rat granulosa cells.Fertil Steril. 2010; 93: 2532-2537Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Our study evaluated the effect of the autophagic process on apoptotic cell death in rat granulosa cells to determine the regulating mechanism of autophagy and apoptosis. Granulosa cells were obtained from 21-day-old female Sprague-Dawley (SD) rats treated with gonadotropin. All animal experimentation was reviewed and approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University School of Medicine. The collection of granulosa cells performed as previous described elsewhere. Briefly, immature female SD rats, 21 to 23 days old, were injected IP with 15 IU of pregnant mare serum gonadotropin (PMSG; Sigma Chemical, St. Louis, MO) to induce ovarian follicular development (20Dhanasekaran N. Moudgal N.R. Biochemical and histological validation of a model to study follicular atresia in rats.Endocrinol Exp. 1989; 23: 155-166PubMed Google Scholar, 21Hughes Jr., F.M. Gorospe W.C. Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia.Endocrinology. 1991; 129: 2415-2422Crossref PubMed Scopus (457) Google Scholar). All rats were killed by cervical dislocation 2 days after gonadotropin treatment, and the ovaries were excised. The granulosa cells were harvested from developing follicles by follicle puncture using a 25-gauge needle (22Rao I.M. Mills T.M. Anderson E. Mahesh V.B. Heterogeneity in granulosa cells of developing rat follicles.Anat Rec. 1991; 229: 177-185Crossref PubMed Scopus (37) Google Scholar). After follicle puncture, the cells were cultured in serum-starved conditions to induce apoptotic cell death. The serum-starved granulosa cells were treated with 3-methyladenine (3-MA; 10 mM; Sigma Chemical) and bafilomycin A1 (Baf A1; 0.1 μM; Sigma Chemical) for inhibiting and promoting the accumulation of autophagosomes, respectively: 3-MA inhibits the formation of autophagosomes by inhibiting the activity of class III PI3K, which is involved in the formation of autophagosomes (23Edinger A.L. Thompson C.B. Death by design: apoptosis, necrosis and autophagy.Curr Opin Cell Biol. 2004; 16: 663-669Crossref PubMed Scopus (1128) Google Scholar, 24Eskelinen E.L. Doctor Jekyll and Mister Hyde: autophagy can promote both cell survival and cell death.Cell Death Differ. 2005; 12: 1468-1472Crossref PubMed Scopus (79) Google Scholar); and Baf A1 causes an accumulation of autophagosomes by reducing the removal of autophagosomes by fusion with lysosomes (25González-Polo R.A. Boya P. Pauleau A.L. Jalil A. Larochette N. Souquère S. et al.The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death.J Cell Sci. 2005; 118: 3091-3102Crossref PubMed Scopus (449) Google Scholar, 26Boya P. González-Polo R.A. Casares N. Perfettini J.L. Dessen P. Larochette N. et al.Inhibition of macroautophagy triggers apoptosis.Mol Cell Biol. 2005; 25: 1025-1040Crossref PubMed Scopus (1448) Google Scholar). The accumulation of autophagosomes was evaluated by the subcellular localization of endogenous microtubule-associated light-chain protein 3 (LC3), which is widely used as a marker of autophagosomes, through immunofluorescence staining using anti-LC3 rabbit polyclonal antibody (diluted 1:500; Novus) and transmission electron microscopy (TEM). Granulosa cell death was examined by Trypan-blue staining. Apoptosis was evaluated by Hoechst 33342 staining and the expression of apoptosis associated protein (bax, bcl-2, cleaved caspase -9 and -3). Granulosa cell death and apoptosis were reported as the mean ± standard error of the mean (SE) of five independent experiments. Statistical analysis was performed by analysis of variance (ANOVA). Significant differences between treatment groups were determined by Duncan's multiple range tests. P<.05 was considered statistically significant. During autophagy induction, LC3 is converted from LC3-I to LC3-II during autophagy. Then LC3-II is localized to isolated membranes and autophagosomes (27Petiot A. Ogier-Denis E. Blommaart E.F. Meijer A.J. Codogno P. Distinct classes of phosphatidylinositol 30-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells.J Biol Chem. 2000; 275: 992-998Crossref PubMed Scopus (1038) Google Scholar, 28Backer J.M. The regulation and function of class III PI3Ks: novel roles for Vps34.Biochem J. 2008; 410: 1-17Crossref PubMed Scopus (515) Google Scholar), and the amount of LC3-II correlates with the number of autophagosomes (29Gagliardi S. Rees M. Farina C. Chemistry and structure activity relationships of bafilomycin A1, a potent and selective inhibitor of the vacuolar H+-ATPase.Curr Med Chem. 1999; 6: 1197-1212PubMed Google Scholar). LC3-I exhibits diffuse expression within the cytoplasm as well as the nucleus, but LC3-II is associated with punctate structures in the cytoplasm (8Choi J.Y. Jo M.W. Lee E.Y. Yoon B.K. Choi D.S. The role of autophagy in follicular development and atresia in rat granulosa cells.Fertil Steril. 2010; 93: 2532-2537Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Therefore, we measured the subcellular localization of endogenous LC3 by immunofluorescence staining to evaluate the accumulation of autophagosomes. As shown in Figure 1A , the punctate structures of LC3-II were present in the cytoplasm of granulosa cells cultured in serum-starved conditions (see Fig. 1A, top) but progressively accumulated in the cytoplasm after Baf A1 treatment (see Fig 1A, bottom). Conversely, in granulosa cells treated with 3-MA, LC3-I appeared to diffuse throughout the cytoplasm (see Fig. 1A, middle).Figure 1(A) Representative immunofluorescence images of granulosa cells cultured in serum-starved conditions. LC3-II was associated with punctate structures in granulosa cells cultured in serum-starved conditions alone (SC, top), but LC3-I had a diffuse expression pattern in the serum-starved granulosa cells treated with 3-methyladenine (3-MA, middle). The presence of punctate structures of LC3-II progressively increased in the serum-starved granulosa cells treated with bafilomycine A1 (Baf A1, bottom). (B) Transmission electron microscopic images of granulosa cells cultured in SC (left), SC + 3-MA (middle) and SC + Baf A1 (right). Arrows indicate representative autophagic vacuoles. N = Nucleus. (C, D) The percentages of dead and apoptotic cells in cultured rat granulosa cells under SC with/without 3-MA or BAf A1. The bar graph represents the mean ± standard error of results from five replicate experiments. ∗Statistically significant difference, P<.05. (E) Representative immunoblots of bax, bcl-2, and cleaved caspase-9 and caspase-3 proteins. In parts C and D, the bar graph represents the mean ± standard error of results from five replicate experiments. ∗Statistically significant difference, P<.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition, we also examined the accumulation of autophagosomes by TEM, which is currently the standard method of detecting autophagosomes in the cell (30Shacka J.J. Klocke B.J. Roth K.A. Autophagy, bafilomycin, cell death: the “a-Bcs” of plecomacrolide-induced neuroprotection.Autophagy. 2006; 2: 228-230Crossref PubMed Scopus (94) Google Scholar). Autophagosomes are characterized by double membranous vacuoles engulfing cytoplasmic materials. Autophagosomes were observed in the serum-starved granulosa cells (see Fig. 1B, left), but the amount decreased in granulosa cells treated with 3-MA (see Fig. 1B, middle). In contrast, numerous autophagosomes were seen in the cytoplasm when granulosa cells were treated with Baf A1 (see Fig. 1B, right). Therefore, it was confirmed that the serum starvation-induced autophagosome accumulation in granulosa cells was decreased by 3-MA but was progressively increased by Baf A1. Under these conditions, the rates of cell death and apoptosis in the Baf A1-treated granulosa cells were statistically significantly higher than those of granulosa cells treated with 3-MA (P<.05; see Fig. 1C and D). These data indicate that the accumulation of autophagosomes induces apoptotic granulosa cell death. However, rates of cell death and apoptosis in granulosa cells treated with serum starvation alone were not higher than those of 3-MA-treated granulosa cells (see Fig. 1C and D), although autophagosomes were observed to accumulate within the cytoplasm of serum-starved cells. This finding suggests that autophagosome accumulation induced by serum starvation by itself may not be sufficient to promote apoptotic granulosa cell death. Similarly, previous studies also reported that apoptotic cell death was promoted through the accumulation of a sufficient amount of autophagosomes induced by prolonged serum starvation and lysosomal inhibition in HeLa cells (18Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. et al.LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5554) Google Scholar, 19Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation.J Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1144) Google Scholar). Taken together, these findings suggest that a certain level of autophagosome accumulation may be needed to promote granulosa cell death and apoptosis. To examine the apoptotic pathway of granulosa cell apoptosis induced by autophagosome accumulation, we evaluated the expression levels of mitochondria-dependent apoptosis proteins. The expression levels of bax and bcl-2 did not change in 3-MA-treated granulosa cells, but bcl-2 expression was decreased in the Baf A1-treated granulosa cells, although bax expression did not change (see Fig. 1E). These findings indicate that the down-regulation of bcl-2 expression was clearly involved in the granulosa cell apoptosis induced by autophagosome accumulation, because the ratio of bax to bcl-2 expression within a cell determines whether a cell will undergo apoptosis or survive (31Oltavi Z.N. Milliman C.L. Korsmeyer S.J. Bcl-2 heterodimerizes in vivo with a conserved homolog. Bax that accelerates programmed cell death.Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5894) Google Scholar). In addition, we evaluated the protein expression of the active forms of capase-9 (initiator caspase) and caspase-3 (executioner caspase) to examine the final step of the apoptosis cascade. The expression levels of cleaved caspase-9 and caspase-3 proteins were statistically significantly increased with autophagosome accumulation induced by Baf A1. These results suggest that the accumulation of autophagosomes increased granulosa cell apoptosis via a decrease in bcl-2 expression and subsequent caspase activation. Our results indicate that the accumulation of autophagosomes induces apoptotic cell death of granulosa cells through decreased bcl-2 expression followed by caspase activation.