Apoptosis of rat ovarian granulosa cells

n granulosa cells by ro and its relevant ca me n ab . It an se p, uld B, HD ssi b histopathology of the testis were observed following inhalation the number of growing oocytes and a shortening of fertile life of Research Article Accepted: 26 November 2011 Published online in Wiley Online Library exposure to n-hexane (De Martino et al., 1987; Nylen et al., 1989; Cao et al., 2007). A two-generation reproductive toxicological study revealed that exposure of rats to commercial hexane for two genera- tions resulted in reduced body weight gains in both F1 and F2 litters at 9000ppm, but no adverse effects on reproduction(Daughtrey et al., 1994). However, it is noted that females account for the great majority of those coming into contact with n-hexane occupationally. Moreover, n-hexane exposure can result in a series of oxidative damage in the ovary of Sprague–Dawley rats (Cao et al., 2007). Epidemiological studies also show that n-hexane and hexane isomers, toluene and methyl ketone, can lead to amenorrhea, menstrual disorder and reduced fertility (Sallmen et al., 2008). Never- theless, its female reproductive toxic mechanisms remains unclear. female Swiss CD1 mice (Siracusa et al., 1992). Ovarian granulosa cells are essential for development of the oocytes. In addition, the apoptosis of ovarian granulosa cells directly or indirectly results in the reduction of germ cell, luteolysis, follicular atresia and finally loss of ability to have children (Cao, 2008). Hence, there *Correspondence to: Wenchang Zhang, Key Laboratory of Environment and Health, Fujian Medical University, Fuzhou, People’s Republic of China. E-mail: wenchang2008@126.com a Key Laboratory of Environment and Health, Fujian Medical University, Fuzhou, People’s Republic of China Keywords: HD; ovarian granulosa cell; apoptosis; gene; n-hexane INTRODUCTION n-Hexane is a volatile organic solvent widely used in industries such as printing, electron device cleaning, mucilage glue, shoe-making and furniture. n-Hexane is identified as a highly dangerous toxicant owing to its high volatility, high fat solubility and cumulative effect (Yan et al., 2011). Previous studies have demonstrated that n-hexane can cause the toxic effects in the central and peripheral nerve system in exposed workers (Puri et al., 2007; Huang, 2008; Misirli et al., 2008; Sendur et al., 2009). There is also some evidence that it can induce toxicological effects in the male reproductive system. Abnormal sperm and varying degrees of severity in the n-hexane exposure and is a useful indicator in evaluating risk of ex- posure to n-hexane (Saito et al., 1991; dos Santos et al., 2002; Prieto et al., 2003). Prieto et al. monitored total 2,5-hexanedione in the urine of 132 Spanish shoe workers who were exposed to n-hexane ranging from 4 to 709mgm�3 (1–200ppm). The concentrations of total urinary 2,5-hexanedione ranged from 0.3–32.46mg l�1 (Prieto et al., 2003). At present, the research with respect to the toxicity of HD mainly limited to neurotoxicity (Opanashuk et al., 2001; Hernandez-Viadel et al., 2003) and testicular toxicity (Blanchard et al., 1996; Boekelheide and Schoenfeld, 2001), while the studies about reproductive toxicity in females are still sparse. Siracusa et al. found that 6weeks of treatment with 1.5% HD in drinking water can cause a statistically significant reduction in Apoptosis of rat ovaria 2,5-hexanedione in vit gene expression Wenchang Zhang,a* Lei Huang,a Can and Huiling Huangc ABSTRACT: Although n-hexane is toxic to the ovary, the related cells is thought to be one of the important reasons for ovaria different concentrations of 2,5-hexanedione (HD, a major met apoptosis, and its relevant gene expression was investigated different exposure time could inhibit the viability of ovarian gr of the ovarian granulosa cells increased notably with the increa inhibit themessage RNA (mRNA) expressionof both bcl-2 and xia on that of bcl-xl, while only an HD concentration of 40mol l�1 co and 60mol l�1 could inhibit the protein expression of NF-k theactivation of NF-kB. In summary, our results indicate that occurrence of apoptosis is related to inhibition of protein expre of fasl and bax, and downregulation of mRNA levels of xiap and Received: 19 June 2011, Revised: 25 November 2011, (wileyonlinelibrary.com) DOI 10.1002/jat.2714 Metabolism studies have demonstrated that 2,5-hexanedione (HD) is a major metabolite of n-hexane in the body (Tshala- Katumbay et al., 2009). Investigations show that HD concentration in the urine of exposed workers correlates well with level of J. Appl. Toxicol. 2012 Copyright © 2012 John n Kong,b Jin Liu,a Lingfeng Luoa chanism remains unknown. Apoptosis of rat ovarian granulosa toxicity. Rat ovarian granulosa cells were exposed in vitro to olite of n-hexane; 0, 20, 40 and 60mmol l�1) to observe the was found by MTT assay that different exposure doses and ulosa cells. After a 12h exposure, we found the apoptosis rate of HD dose. The HD concentration of each exposure dose could and increase themRNAexpression of fasl without any influence raise themRNA expression of bax. Concentrations of HD of 40 but only an HD concentration of 60mol l�1 could improve can induce the apoptosis of ovarian granulosa cell, and the on of NF-kB, activation of NF-kB, upregulation of mRNA levels cl-2. Copyright © 2012 John Wiley & Sons, Ltd. b The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China c Union Hospital of Fujian Medical University, Fuzhou, People’s Republic of China Wiley & Sons, Ltd. MATERIALS AND METHODS Rats were subcutaneously injected with pregnant mare serum Cell viability following HD exposure was examined using an 3-(4,5- W. Zhang et al. are reasons to suspect that the apoptosis of ovarian granulosa cells could be one of themechanisms of n-hexane-induced toxic effects in the female reproductive system. Nevertheless, the question remains whether exposure in vitro of HD can affect apoptosis in ovarian granulosa cells. The apoptosis of the ovarian granulosa cells is mediated by various factors (Li et al., 2007). Bcl-2, Bcl-xl and Xiap are genes that inhibit cell apoptosis, while Bax and FasL are genes that promote cell apoptosis. These genes can activate a cascade of events relating to apoptosis through different pathways (Holcik et al., 2001; Morizane et al., 2005; Chen et al., 2005; Slot et al., 2006; Voss et al., 2008). The major players of apoptosis in the ovary are assumed to be the Fas system and the Bcl-2 family members. Fas receptor (Fas/CD95) is a member of the tumor necrosis factor/nerve growth factor family, which is activated upon binding Fas ligand, leading to receptor aggregation and formation of a death-inducing signaling complex (DISC). Once Fas has been activated, two distinct intracellular apoptotic pathways can be executed, depending on the level of released active caspase-8. High levels of caspase-8 at the DISC directly initiate cleavage of other downstream effector caspases, such as caspase-3, thereby initiating the execution phase of apoptosis. However, a small caspase-8 signal is generated, which in order to induce apoptosis, requires an amplification loop via the mito- chondria involving cleavage of the pro-apoptotic Bcl-2 family member Bid. It has been proposed that cleaved Bid stimulates dimeric binding of other pro-apoptotic Bcl-2 family members (e.g. Bax and Bak) to the mitochondria at the expense of anti- apoptotic Bcl-2 family members (e.g. Bcl-2, Bcl-w, Bcl-xl). This triggers cytochrome c release, which eventually results in down- stream activation of effector caspases and cell death (Slot et al., 2006). The rate of expression levels between apoptosis-inhibition genes and apoptosis-promotion genes determines largely whether cell apoptosis will occur or not. However, it remains unknown that which genes are related to the induction of apo- ptosis by HD. A better understanding of the expression of these apoptosis relevant genes may help to delineate the mechanisms of n-hexane-induced toxic effects. A recent report (McDermott et al., 2008) showed the n-hexane toxicity in Jurkat T-cells is mediated by reactive oxygen species (ROS). Increased ROS production and leakage of lactate dehydro- genase (LDH) have been reported in murine neural progenitor cells (NPC) treated with HD for 24 h (0.5–50mM; Kim et al., 2009). It has been demonstrated by several experiments that influences on the activity of NF-kB by ROS vary in different types of cells (Korn et al., 2001; Storz et al., 2004; Li and Peng, 2008), while ROS in the ovarian follicular fluid mainly derives from ovar- ian granulosa cells (Kodaman and Behrman, 2001; Seino et al., 2002). Moreover, Xiap, Bcl-2, Bcl-xl and FasL are all the target genes regulated by nuclear factor-kappa B (NF-kB) which has been proved closely related to cell apoptosis (Dutta et al., 2006). Taken together, it is possible to infer that NF-kB plays a vital role in the toxic effects by HD. The usual NF-kB refers to dipolymer p50/p65, since it has been found to be a transcrip- tionally active NF-kB of the widest distribution, richest content, and strongest biological activity. We hypothesized that apoptosis of rat ovarian granulosa cells might be involved in n-hexane-induced ovarian toxicity. Therefore, in the present study, we directly exposed rat ovarian granulosa cells in different concentrations of HD in vitro, in order to evaluate the apoptotic effects and its possiblemechanism.We examined the cell viability and the cell apoptosis, detected the corresponding Copyright © 2012 Johnwileyonlinelibrary.com/journal/jat Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as- say. Ovarian granulosa cells cultured in 96-well plates after 0, 12, 24 and 36h exposure were washed twice with PBS, and 200ml MTT solution (0.5mg ml�1 in PBS) was added to each well for 4 h, followed by 100ml solubilization solution (dimethyl sulfoxide and ethanol, 1 to 1 ratio) to lyse the cells and solubilize the MTT formazan crystals formed. The optical density (OD) was read at 490 nm using the enzyme immunoassay instrument. Untreated negative controls were run together with the treated cells, and plates with culture media only served as background controls. After background OD subtraction, the results were expressed as a percentage of the average negative control. The tests were repeated three times and six repeat wells were designed. TUNEL Assay–Apoptosis Index Apoptosis of ovarian granulosa cells after a 12 h exposure was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) fluorescent staining with the detecting kit (Jiangsu Biyuntian Biological Technology Institute), following the protocol given with the kits. Antifade gonadotropin (PMSG, 10 IU) to stimulate follicular development. At 48 h after PMSG injection, ovaries were collected and punc- tured to isolate granulosa cells (Zhou et al., 2008). Granulosa cells were washed and centrifuged (1000g, 10min). Next, the cells were plated at a density of 1� 106 cells ml�1 in six-well culture plates (1.5ml per well) or 96-well culture plates (200 ml per well) or glass bottom cell culture dish (100 ml per well) and cultured in Dulbecco’s modified Eagle medium, Nutrient Mixture F-12 (DMEM/F-12), containing 10% fetal bovine serum at 37 �C in 5% CO2 . Following the 24 h acclimation period, the medium was replaced with cell culture media containing HD (final concentration 0, 20, 40, and 60mmol l�1, respectively). The cells normally cultured (HD of 0mmol l�1) were used as control. MTT Assay Reagents and Animals 2,5-Hexanedione was purchased from Sigma (St Louis, MO, USA) and its purity was greater than 98%. Forty-five female Wistar rats (21days old) were purchased from Shanghai Slac Laboratory Animal Co. Ltd [clean level, animal license number SCXK(Hu) 2007-0005]. All rats were acclimated for 1week prior to use and maintained in a controlled environment with free access to water and food, under a 12h light–dark cycle and at a constant tempe- rature of 23� 2 �C. All animal experimental procedures were approved by the Institutional Animal Ethics Committee and performed according to the guidelines for animal welfare at Fujian Medical University. Preparation and Exposure of Ovarian Granulosa Cell mRNA expression of apoptosis relevant genes, including Bcl-2, Bcl-xl, Xiap, FasL and Bax. Also, we detected the location and the fluorescence intensity of P65 to analyze the expression and the activity of NF-kB. J. Appl. Toxicol. 2012Wiley & Sons, Ltd. with Fluorescein isothiocyanate (FITC) was added, with PBS for rinsing after approximately 45min of incubation at room tempera- ture and in dark place. Propidium iodide (PI) was added for restain- ing for 30min. Distribution of nuclear translocation of NF-kB p65 protein was observed using a laser scanning confocal microscopy (LSCM; SP5, Leica) after PBS rinse, with an excitation wavelength of 488nm and an emission wavelength of 560nm. The intensities sented translocation and activation level of p65 protein. Amplified fragment length 5′-GGAGATTACTGCCCTGGCTCCTA-3′ 150 bp 5′- GACTCATCGTACTCCTGCTTGCTG-3′ 5′- AAGATCAGCCTGTGGCATCTTTG-3′ 104 bp 5′- GCTCCCATAGCTGCGCTGTAA-3′ 5′- TGAACCGGCATCTGCACAC-3′ 116 bp 5′- CGTCTTCAGAGACAGCCAGGAG-3′ 5′- CACTTTCCCAACTGCTTCTTC-3′ 144bp 5′- AGTAACGATCCGTGCGTCATA-3′ 5′- CACCAACCACAGCCTTAGAGTATCA-3′ 171 bp 5′- ACTCCAGAGATCAAAGCAGTTCCA-3′ 5′-TTGTTACAGGGTTTCATCCAGG-3′ 180 bp 5′-CGTGTCCACGTCAGCAATCA-3′ Apoptosis and disruption of relevant gene expression by HD PCR Assay After a 12 h exposure, the total RNA was isolated from granulosa cells using Trizol (Invitrogen). Synthesis of first-strand cDNA was performed by reverse transcription using PrimeScript RT Reagent Kit (TaKaRa) according to the manufacturer’s protocol. The primers used to amplify the cDNA sequences are shown in Table 1 (Takara Company). The specificity for each primer set was confirmed by analyzing the melting(dissociation) curve under the optimized conditions (95 �C for 15 s) using a 7500 Real-Time PCR System (Applied Biosystems, USA) after each real-time PCR. The reactions were set up with 10ml of SYBR Premix Ex Taq, 0.8ml of primer mixture (10mM), 0.4ml of ROX Reference Dye II, mounting medium was used to cover the slice. An inverted fluo- rescence microscopy (DFC-480, Leica) was used to observe the results and the excitation wavelength was 488 nm. Six fields of vision were randomly selected for each well on the six-well plate, and 200 cells were randomly counted for each field of vision. Un- der fluorescence microscopy, the cytoplasm of all cells was found to be uniform pale green, while the nucleus of the apo- ptotic cells were shown as dense and heavily stained dark green fluorescence lightspots. The total apoptotic cell number and the total cell number were counted for six fields of vision. The ratio of the two numbers was the apoptosis rate of granulosa cells, namely, apoptosis index (AI), for the corresponding well. FasL Forward primer Reverse primer Bax Forward primer Reverse primer Table 1. Primer sequences Gene Primer sequences b-acting Forward primer Reverse primer Bcl-xl Forward primer Reverse primer Bcl-2 Forward primer Reverse primer Xiap Forward primer Reverse primer 2.0ml of cDNA template and 6.8ml of dH2O under the following optimized conditions: 60 �C for 34 s followed by 45 cycles. Relative mRNA levels were normalized to the endogenous reference gene b-actin and calculated using the 2-ΔΔCT method as described previously (Livak and Schmittgen, 2001; Liu et al., 2009). Immunofluorochemistry Assay After a 12 h exposure, the ovarian granulosa cells cultured in the glass bottom cell culture dish were rinsed with PBS, then fixed with 95% ethanol at 20 �C for 30min. Rupture of membranes was done by 0.2% Triton X-100. A 100ml aliquot of goat serum- confining liquid was added for incubation at room temperature for 15min. The serum-confining liquid was removed and a 1:200 dilution of the first antibody (rabbit antirat polyclonal antibody NF-kB p65, sc-109, Santa Cruz) was added and placed in a refrig- erator at 4 �C for one night. PBS was used for rinsing after 30min of rewarming and 1:200 dilution of the second antibody labeled J. Appl. Toxicol. 2012 Copyright © 2012 John Statistics The experimental data were expressed as means� SD. One-way analysis of variance (ANOVA) and least significant difference of green fluorescence of cytoplasm area, nucleus area and total cell were recorded by an image processing software to represent protein level of p65, respectively, and the intensity ratio (Fc/Fn) of green fluorescence of cytoplasm area and nucleus area repre- Figure 1. Impact of exposure time and doses of 2,5-hexanedione on the viability of rat ovarian granulosa cells. Cell viability was assessed as de- scribed in theMaterials andMethods section and expressed asmeans� SD. Significant difference: (b) P< 0.05 compared with all groups at time zero, the control, 20 and 40mmol l�1 groups at 12h and the control group at 24h; (c) P< 0.05 compared with all groups at time zero, 12 h and 36h, and the 40mmol l�1 group at 24h; (d) P< 0.05 compared with all groups at time zero, 12h and 24h, and the control, 40 and 60mmol l�1 groups at 36h; (e) P< 0.05 compared with all groups at time zero, 12 h and 24h, and the control and 20mmol l�1 groups at 36h. Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat W. Zhang et al. (LSD) tests were carried out to determine significant differ- ences of multiple comparisons (SPSS program, version 13.0). A P-value of < 0.05 was considered statistically significant. Figure 2. Cells of each group of the same field of vision under common o exposure (A–D). The arrow indicates typical apoptotic cells. As the exposure d became more and more intense. (A) Cells treated with 0mmol l�1 HD for 1 40mmol l�1 HD for 12 h. (D) Cells treated with 60mmol l�1 HD for 12 h. Copyright © 2012 Johnwileyonlinelibrary.com/journal/jat The interaction between different indexes and doses and times were analyzed by Spearman correlation (correlation coefficient was expressed as r). ptical microscopy (left) and fluorescence microscopy (right) after a 12 h ose of 2,5-hexanedione (HD) rose, the dark green fluorescence lightspots 2 h. (B) Cells treated with 20mmol l�1 HD for 12 h. (C) Cells treated with J. Appl. Toxicol. 2012Wiley & Sons, Ltd. instead of cell death. After exposure for 24 h, the cell viability HD increased (r =�0.623, P< 0.01), which indicated that HD showed no significant difference compared with the control group (P< 0.05), which indicated that only a certain range of exposure doses of HD could activate NF-kB by relatively improving the translocation level of p65 protein. DISCUSSION Some previous in vitro and in vivo reports using HD over the millimolar dose ranges have shown a variety of toxic effects on cellular organelles and processes (Heijink et al., 2000; LoPachin and DeCaprio, 2004). HD (20–60mM) can induce injury and apo- ptosis to rat pheochromocytoma cells (PC12) after exposure for 24 h (Sun et al., 2005). After exposing human neuroblastoma SK-N-SH cells to HD for 48 h, the cell viability fell precipitously over the range 10–30mM, with an IC50 of 22.4� 0.2mM, and there was a significant increase in apoptotic nucleoids over the range 8.5–17mM (Zilz et al., 2007). A recent study suggested that some relatively low doses of HD (0.5–50mM) can also dose- dependently suppress NPC proliferation and cell viability (Kim et al., 2009). In the present study, we exposed ovarian granulosa cells to relatively high doses of HD. In MTT assay, we found that the cell viability in every group diminished as the exposure dose section and expressed as means� SD from six replicate wells. Significant Apoptosis and disruption of relevant gene expression by HD Detection of Apoptosis Index The cells with dense and heavily stained dark green fluorescence lightspots were