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FASL-mediated cell death in the bovine endometrium
Animal Reproduction Science 151 (2014) 97–104
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FAS/FASL-mediated cell death in the bovine endometrium夽 Miki Arai a , Shin Yoshioka a , Ryo Nishimura b , Kiyoshi Okuda a,∗ a Laboratory of Reproductive Physiology, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan b Laboratory of Theriogenology, Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8550, Japan
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Article history: Received 4 June 2014 Received in revised form 26 September 2014 Accepted 5 October 2014 Available online 14 October 2014 Keywords: Cattle Endometrium Remodeling Apoptosis
a b s t r a c t In many mammals, endometrial cells are remodeled by apoptosis and cell proliferation throughout the estrous cycle. Although apoptosis is known to be induced by various factors involving two major apoptotic pathways (the death receptor- and mitochondria-mediated pathways), how it is regulated in the bovine endometrium is unclear. We examined (1) the cyclic expressions of apoptosis-related factors, FAS, DcR3, BCL2 and BAX, in the bovine endometrium and (2) the effect of death ligands on the viability of, and FAS mRNA expression in, cultured bovine endometrial epithelial and stromal cells. FAS expression did not change during the estrous cycle, whereas DcR3 expression was higher at the mid and late luteal stages than at the early luteal and follicular stages. BCL2 expression was higher at the late luteal stage than at the early luteal and follicular stages, and the BAX/BCL2 ratio was higher at the early luteal stage than at the late luteal stage. Treatment or pretreatment with tumor necrosis factor-␣ (TNF) + interferon ␥ (IFNG) in combination with FAS ligand significantly reduced the viability of both epithelial and stromal cells. Furthermore, TNF + IFNG treatment significantly increased the expression of FAS mRNA in both types of endometrial cells. The overall results suggest that both extrinsic and intrinsic pathways are involved in remodeling the bovine endometrium throughout the estrous cycle, and that the death ligands produced by immune cells and the endometrium play important roles in inducing cyclic endometrial cell death. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In many mammals, endometrium changes morphologically and functionally during the estrous cycle, and is remodeled by cyclic cell proliferation and apoptosis (Johnson et al., 1997; Okano et al., 2007; Roberto da Costa et al., 2007; Wood et al., 2007). Recently, we demonstrated that both cell proliferation and apoptosis (programmed cell
夽 This research was supported by a Grant-in-Aid for Scientific Research (No. 24380155) of the Japan Society for the Promotion of Science (JSPS). ∗ Corresponding author. Tel.: +81 86 251 833; fax: +81 86 251 8349. E-mail address: [email protected] (K. Okuda). http://dx.doi.org/10.1016/j.anireprosci.2014.10.004 0378-4320/© 2014 Elsevier B.V. All rights reserved.
death) are active in the bovine endometrium at the follicular and early luteal stages (Arai et al., 2013). Apoptosis has critical roles in the development and functional maintenance of a variety of tissues including the human endometrium (Kerr et al., 1972; Strasser et al., 2000; Harada et al., 2004; Jin and El-Deiry, 2005). Apoptotic signaling occurs through two major pathways, the death receptor-mediated (extrinsic) pathway and the mitochondria-mediated (intrinsic) pathway (Nagata, 1997; Jin and El-Deiry, 2005). The extrinsic apoptosis pathway is mainly regulated by ligands and receptors of the tumor necrosis factor superfamily, which are referred to as “death ligands” and “death receptors” (Strasser et al., 2000; Jin and El-Deiry, 2005). Tumor necrosis factor-␣ (TNF) is a cytokine
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that participates in various physiological and inflammatory responses by binding to TNF receptor (TNFR) 1 and TNFR2 at the cell membrane (Nagata, 1997). TNFR1 has a cytoplasmic death domain and induces not only apoptosis, but also the activation of NF-B (Nagata, 1997). In contrast, TNFR2 lacks a death domain, and is suggested to modulate the actions of TNFR1 (Van Herreweghe et al., 2010). FAS, a TNF superfamily receptor, is known to induce apoptotic cell death when it binds to FAS ligand (FASL) (Jin and El-Deiry, 2005), which is mainly expressed in activated T lymphocytes and natural killer (NK) cells (Nagata, 1999). The binding of ligands to TNFR1 or FAS recruits and activates a series of cysteine proteases, called caspases (Jin and El-Deiry, 2005). The binding induces caspase-8 activation through the adaptor protein Fas-associated death domain and then induces apoptosis by means of activation of caspase-3 (Jin and El-Deiry, 2005). FASL binds not only to FAS but also to decoy receptor 3 (DcR3), which is a soluble decoy receptor that lacks a transmembrane domain and that neutralizes FAS-induced apoptosis (Pitti et al., 1998; Lin and Hsieh, 2011). The mitochondrial pathway is mainly regulated by BCL2 family proteins (Youle and Strasser, 2008). BCL2 family members are categorized into three groups based on their functions and structures: (1) pro-survival BCL2 family members (e.g., BCL2, BCL-XL), (2) pro-apoptotic BCL2 family members (e.g., BAX, BAK) and (3) proapoptotic BH3-only proteins (e.g., BID, BAD, BIM) (Youle and Strasser, 2008). The pro-apoptotic BAX permeabilizes the outer mitochondrial membrane and releases cytochrome c (Youle and Strasser, 2008). Cytochrome c released into the cytosol binds to apoptotic proteaseactivating factor-1 (APAF1) and then activates caspase-9 to turn on caspase-3 (Youle and Strasser, 2008). By contrast, the anti-apoptotic BCL2 inhibits BAX activation and the subsequent cytochrome c release (Oltvai et al., 1993; Jin and El-Deiry, 2005). Therefore, the ratio of BAX to BCL2 is thought to be important for decision of cellular survival (Oltvai et al., 1993). Activation of caspase8 in the death receptor-mediated pathway activates a pro-apoptotic BH3-only protein, BID, which promotes apoptosis via the mitochondrial pathway (Youle and Strasser, 2008). Some members of TNF and TNFR superfamilies and BCL2 family are expressed in the endometrium throughout the ovarian cycle in cattle (Okuda et al., 2010), cat (Liman et al., 2013), monkey (Wei et al., 2005), marmoset (Narkar et al., 2006) and human (Hunt et al., 1992; Philippeaux and Piguet, 1993; Watanabe et al., 1997; Yamashita et al., 1999; Tao et al., 1997; Vaskivuo et al., 2000). These include TNF (Hunt et al., 1992; Philippeaux and Piguet, 1993), FAS (Watanabe et al., 1997; Yamashita et al., 1999; Wei et al., 2005), DcR3 (Chen et al., 2009) and BCL2 and BAX (Tao et al., 1997; Vaskivuo et al., 2000; Wei et al., 2005; Narkar et al., 2006; Liman et al., 2013). In many of those species, cyclic endometrial apoptosis is controlled by periodic expressions of apoptosis regulatory factors (Harada et al., 2004). In the endometrium of human and other primates, BAX expressions are high during the secretory (luteal) phase (Tao et al., 1997; Wei et al., 2005; Narkar et al., 2006), while the changes in BCL2 expression during the menstrual cycle
are not consistent among reports (Watanabe et al., 1997; Tao et al., 1997; Yamashita et al., 1999; Narkar et al., 2006). Serum DcR3 levels are high during the mid luteal phase and during normal pregnancy in the human endometrium (Chen et al., 2009), while FAS protein expression does not change throughout the menstrual cycle (Watanabe et al., 1997; Yamashita et al., 1999). We recently reported that the bovine endometrium is also remodeled throughout the estrous cycle (Arai et al., 2013). In the late luteal and the follicular stages in the bovine endometrium, increases are observed in TNF mRNA levels (Okuda et al., 2010) and in the numbers of T lymphocytes and NK cells, both of which produce FASL, TNF and interferon ␥ (IFNG) (Cobb and Watson, 1995; Oliveira et al., 2013). However, it is unclear how the expressions of other pro- and anti-apoptotic factors in the bovine endometrium during the estrous cycle are involved in regulating endometrial apoptosis. To understand how apoptosis is regulated during remodeling of bovine endometrium, we examined (1) cyclic expressions of FAS, DcR3, BCL2 and BAX in the bovine endometrium and (2) the effects of death ligands (TNF, IFNG and FASL), on the viability of, and FAS mRNA expression in, cultured bovine endometrial epithelial and stromal cells. 2. Materials and methods 2.1. Collection of endometrial tissues Apparently healthy uteri of Holstein cows without a visible conceptus were obtained within 10–20 min after exsanguinations and were immediately transported to the laboratory. Stages of the estrous cycle were confirmed by macroscopic observation of the ovary and uterus as described previously (Okuda et al., 1988; Miyamoto et al., 2000). For protein determination, endometrial tissues (n = 4 cows/stages) were collected from cows at four different stages of the estrous cycle (estrus = Day 0; early luteal: Days 2–3; mid luteal: Days 8–12; late luteal: Days 15–17 and follicular stage: Days 19–21). Intercaruncular endometrial tissues from the uterine horn, ipsilateral to the CL or the dominant follicle, were used for experiments. Endometrial tissues were immediately frozen in liquid nitrogen, and stored at −80 ◦ C until processed for protein isolation. For cell culture experiments, the uterus was submerged in ice-cold physiological saline and transported to the laboratory within 1–1.5 h on ice. 2.2. Isolation and culture of endometrial cells Uteri at Days 2–5 after ovulation were utilized for cell culture. Epithelial and stromal cells from bovine endometrium were separated as described previously (Murakami et al., 2003; Arai et al., 2013). After the collecting epithelial cells by incubating with trypsin [0.3% (w/v) (>7500 BAEE units/mg solid; T9201; Sigma-Aldrich, St. Louis, MO, USA)], stromal cells were isolated by dissociation of minced endometrial tissues by incubating with collagenase [0.05% (w/v); no. CLS1; Worthington Biochemical Co., Lakewood, NJ, USA] and DNase I [0.005% (w/v); DNP2; BBI Enzymes, Cardiff, UK]. The isolated epithelial
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and stromal cells were counted using a hemocytometer, and the viability of both cell types was greater than 85% as assessed by 0.5% (w/v) trypan blue dye exclusion. Final pellets of the epithelial and stromal cells were separately resuspended in culture medium (DMEM/F-12, 1:1 Mixture: 12400-024; Life Technologies, Carlsbad, CA, USA) supplemented with 10% (v/v) bovine serum (16170078; Life Technologies), 20 g/ml gentamicin (G1397; Sigma-Aldrich) and 2 mg/ml amphotericin B (A9528; Sigma-Aldrich). The homogeneity of stromal cells and epithelial cells was evaluated using immunofluorescent staining for specific markers of epithelial (cytokeratin) and stromal cells (vimentin) as described previously (Malayer and Woods, 1998; Tanikawa et al., 2008). The epithelial cell contamination of stromal cells was about 1%, and the stromal cells contamination of epithelial cells was <1%. Stromal cells were seeded at a density of 1.0 × 105 viable cells/ml in 96-well cluster dishes (3860-096; Asahi Techno Glass, Chiba, Japan) for cell-viability experiments or 24-well cluster dishes (no. 662160; Greiner Bio-One, Frickenhausen, Germany) for FAS mRNA expression determinations. To purify the stromal preparation, the medium was changed 2 h after plating, by which time selective attachment of stromal cells had occurred (Fortier et al., 1988). Epithelial cells were seeded at a density of 1.0 × 105 viable cells/ml in 75 cm2 culture flasks (no. 658175; Greiner Bio-One) for trypsinization. Since epithelial cells attached 24–48 h after plating, the medium in the epithelial cell culture was replaced 48 h after plating. When epithelial cells reached sub-confluence, the cells were trypsinized to purify the epithelial cells. Briefly, epithelial cells in the culture flask were washed with phosphate-buffered saline (PBS) twice. After the washing, 0.02% (w/v) porcine trypsin (1000–2000 BAEE units/mg solid; Sigma-Aldrich) with 0.008% (w/v) EDTA (SigmaAldrich) in PBS was added to the flask and the cells were incubated for 5 min at 38 ◦ C to detach stromal cells. Then, the solution containing the stromal cells was removed, and the remaining cells were incubated with 2 mmol/l EDTA in PBS for 2 min at 38 ◦ C. After the incubation, the solution was removed and the cells were washed with PBS, and the cells were incubated for 10 min at 38 ◦ C with 0.02% (w/v) bovine trypsin in PBS. After the incubation, the cells were washed by centrifugation (180 × g for 10 min at 4 ◦ C) with culture medium. Then, epithelial cells were seeded at the same density (1.0 × 105 viable cells/ml) as stromal cells, and cultured at 38 ◦ C in a humidified atmosphere of 5% CO2 in air until the cells reached confluence. 2.3. Experiment 1: FAS, DcR3, BCL2 and BAX protein expressions throughout the estrous cycle FAS, DcR3, BCL2 and BAX protein levels in endometrial tissues were assessed by Western blot analysis (n = 4 experiments). Endometrial tissues were homogenized on ice in the homogenization buffer by a tissue homogenizer (NS-50; Physcotron; Niti-on, Chiba, Japan), followed by filtration with a metal wire mesh (150 m). For each protein analysis, tissue homogenates were centrifuged at 600 × g for 10 min and these supernatants were
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recentrifuged at 9000 × g for 30 min. After centrifugation, supernatant were collected as cytoplasmic fractionations for FAS and DcR3 protein analysis and precipitates were collected as mitochondrial fractionations for BCL2 and BAX protein analysis. Protein concentrations were determined by BCA method (Osnes et al., 1993). The proteins were heated with SDS gel-loading buffer containing 1% (v/v) -mercaptoethanol (137-06862; Wako Pure Chemical Industries) at 95 ◦ C for 10 min. Samples (25 g protein) were subjected to SDS-PAGE (15%) for 1.5 h at 200 V. Separated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (RPN303F; GE Healthcare, Milwaukee, WI, USA) for 30 min at 200 mA using a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA). The membrane was washed in TBS with 0.1% Tween 20 (TBST), incubated in PVDF Blocking Reagent for Can Get Signal (NYPBR01; TOYOBO, Osaka, Japan) for 60 min at room temperature, incubated separately with specific primary antibodies to FAS (55 kDa: 05-351; Merck KGaA, Darmstadt, Germany; 1:500 dilution), DcR3 (32 kDa: no. 4758; Cell Signaling Technology, Boston, MA, USA; 1:1000 dilution), BCL2 (28 kDa: no. 2876; Cell Signaling Technology; 1:1000 dilution), BAX (20 kDa: no. 2772; Cell Signaling Technology; 1:1000 dilution) and ACTB (42 kDa: A2228; Sigma-Aldrich; 1:4000 dilution) in Can Get Signal Immunoreaction Enhancer Solution 1 (NKB201; TOYOBO) overnight at 4 ◦ C, incubated with secondary antibodies to FAS (554017; HRP Goat Anti-rat Ig; Becton, Dickinson and Company, Franklin Lakes, NJ, USA; 1:500 dilution), to DcR3, BCL2 and BAX (NA934; antirabbit Ig, HRP-linked whole antibody produced in donkey; GE Healthcare; 1:4000 dilution) and to ACTB (NA931; anti-mouse Ig, HRP-linked whole antibody produced in sheep; GE Healthcare; 1:40,000 dilution) in Can Get Signal Immunoreaction Enhancer Solution 2 (NKB-301; TOYOBO) for 60 min at room temperature, incubated in Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500; Merck) and captured using a ChemiDocTM XRS+ System (Bio-Rad). The intensity of the immunological reaction was estimated by measuring the optical density in the defined area by computerized densitometry using an Image LabTM software (170-8265J1PC; Bio-Rad). 2.4. Experiment 2: Effect of FASL, TNF and IFNG on cell viability When cells were confluent, the culture medium was replaced with DMEM/F-12 without phenol red medium-BSA [DMEM/F-12 without phenol red (D2906; Sigma-Aldrich) supplemented with 0.1% (w/v) BSA, 5 ng/ml sodium selenite (S5261; Sigma-Aldrich), 0.5 mM ascorbic acid (031-12061; Wako Pure Chemical Industries, Ltd., Osaka, Japan), 5 mg/ml transferrin (T4132; SigmaAldrich) and 20 mg/ml gentamicin]. Then, the cells were cultured in the presence or absence of stimulants [TNF (100 ng/ml, HF-13; kindly donated by Dainippon pharmaceutical, Osaka, Japan), IFNG (50 ng/ml, kindly donated by Dr. Inumaru, National Institute of Animal Health, Ibaraki, Japan) and FASL (50 ng/ml, F0427; SigmaAldrich)] for 24 h, after pretreatment with TNF and IFNG for 24 h (n = 3 experiments) or no pretreatment
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(n = 3 experiments). Cell viability was determined by a Dojindo Cell Counting Kit, including WST-1 (no. 34506463; Dojindo, Kumamoto, Japan) as described previously (Komiyama et al., 2008). Briefly, WST-1 [2-(4-iodophenyl)3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H-tetrazolium, monosodium salt], a derivative of MTT (3-[4,5-dimethyl2thiazolyl]-2,5-diphenyl-2H-tetrazolium/Br), is a tetrazolium salt reduced to formazan dye by live cells containing active mitochondria. For the viability assay, the culture medium was replaced with 100 ml DMEM/F-12 medium without phenol red, and then 10 ml of assay solution [0.3% WST-1, 0.2 mM 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS) in PBS, pH 7.4] was added to each well. The cells were then incubated for 3 h at 38 ◦ C. The absorbance (A) was read at 450 nm using a microplate reader (model 450; Bio-Rad). Cell viability (%) was calculated as cell viability (%) = 100 × (Atest /Acontrol ), where Acontrol is the mean A of non-treated wells, and Atest is the mean A of FASL-, TNF + IFNG-, and FASL + TNF + IFNGtreated wells. The mean A of wells in the absence of the cells was subtracted from the mean A of all experimental wells.
2.5. Experiment 3: Effect of TNF and IFNG on FAS mRNA expression When cells were confluent, the culture medium was replaced with DMEM/F-12 without phenol red mediumBSA in the presence or absence of TNF (100 ng/ml) and IFNG (50 ng/ml) for 24 h (n = 4 experiments). After 24 h of culture, total RNA was extracted from endometrial cells using TRIsure (no. BIO-38033; Bioline, London, UK) according to the manufacturer’s directions. Total RNA concentration (g/ml) was calculated as 40 × A260 . The purity of RNA was evaluated by the ratio of A260 /A280 , and samples with ratio 2.0 0.99). To analyze the relative level of expression of each mRNA, the 2−CT method was used (Livak and Schmittgen, 2001).
2.6. Statistical analysis All experimental data are shown as the mean ± SEM. The statistical significance of differences was assessed by analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test using GraphPad Prism ver. 6.0 for Mac (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Experiment 1: FAS, DcR3, BCL2 and BAX protein expression throughout the estrous cycle FAS and DcR3 were expressed in the bovine endometrium throughout the estrous cycle (Fig. 1A and B). FAS expression in the bovine endometrium did not significantly change during the estrous cycle (Fig. 1A), whereas DcR3 expression was higher at the mid and late luteal stages than at the early luteal and follicular stages (Fig. 1B; P < 0.05). BCL2 and BAX protein were also expressed in the bovine endometrium throughout the estrous cycle (Fig. 1C and D). BCL2 expression was lower at the early luteal and follicular stages than at the late luteal stage (Fig. 1C; P < 0.05). BAX expression did not significantly change during the estrous cycle (Fig. 1D). Furthermore, the BAX/BCL2 ratio was higher at the early luteal stage than at the late luteal stage (Fig. 1E; P < 0.05). 3.2. Experiment 2: Effect of FASL and TNF + IFNG on cell viability FASL alone did not affect the viability in epithelial and stromal cells (Fig. 2). Furthermore, TNF + IFNG with or without FASL did not reduce the viability of epithelial cells (Fig. 2A). On the other hand, the viability of stromal cells was decreased to approximately 60% in cultures with FASL in combination with TNF + IFNG (Fig. 2B; P < 0.05). After the pretreatment with TNF + IFNG for 24 h, FASL or TNF + IFNG alone reduced the viability of epithelial cells (Fig. 2C; P < 0.05). TNF + IFNG in combination with FASL reduced the viability of epithelial cells compared with FASL or TNF + IFNG alone (Fig. 2C; P < 0.05). In stromal cells after the pretreatment with TNF + IFNG, TNF + IFNG decreased cell viability (Fig. 2D; P < 0.05). Moreover, FASL with or without TNF + IFNG significantly reduced the viability of stromal cells to less than 30% (Fig. 2D; P < 0.05). 3.3. Experiment 3: Effect of TNF + IFNG on FAS mRNA expression TNF + IFNG significantly increased the expression of FAS mRNA in epithelial and stromal cells (Fig. 3; P < 0.05). 4. Discussion In bovine endometrium, it is unclear whether apoptosis is related to the regulation of its physiological function. Recently, we obtained evidence for apoptosis in bovine endometrium by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase-3
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Fig. 1. Changes in FAS (A), DcR3 (B), BCL2 (C) and BAX (D) protein expressions (relative ratios to ACTB protein expression) and BAX/BCL2 ratio (E) in the bovine endometrium throughout the estrous cycle (mean ± SEM, n = 4 experiments). Different letters indicate significant differences (P < 0.05).
(CCP3) (Arai et al., 2013). We also found that the population of apoptotic endometrial cells changed during the estrous cycle (Arai et al., 2013). In the present study, we confirmed that FAS, one of death receptors, is expressed in bovine endometrium. These findings indicate that apoptosis is an important regulatory mechanism for bovine endometrial function. In addition, studies with cultured endometrial cells showed that cell death was induced by FASL in the presence of TNF and IFNG. This result suggests that FAS/FASL-mediated cell death is related to the cyclic changes in bovine endometrium. FAS-mediated apoptosis participates in the regulation of human endometrial function (Otsuki, 2001; Song et al., 2002; Harada et al., 2004). DcR3, a member of the TNFR superfamily, competes with FAS for binding to FASL and inhibits FASL-induced apoptosis (Pitti et al., 1998), so that it
is necessary to determine the expressions of both FAS and DcR3 to evaluate FASL-induced apoptosis. In the present study, FAS expression did not significantly change during the estrous cycle (Fig. 1A), while DcR3 expression was higher at the mid and late luteal stages than at the early luteal and follicular stages in the bovine endometrium (Fig. 1B). These findings agreed with our previous findings that cleaved caspase-3 expression was higher at the early luteal and follicular stages than at the mid and the late luteal stages in the bovine endometrium (Arai et al., 2013). The number of T lymphocytes and NK cells, which are known to produce FASL, increases in bovine uterus during luteolysis (Cobb and Watson, 1995; Oliveira et al., 2013). The increase of those cells and the decrease of DcR3 expression may induce apoptosis in the bovine endometrium during luteolysis. In human endometrial
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Fig. 2. Effects of FASL and TNF + IFNG on the viability of cultured bovine endometrial cells. (A and C): Epithelial cells without and with 24-h TNF + IFNG pretreatment, respectively. (B and D): Stromal cells without and with 24-h TNF + IFNG pretreatment, respectively. Concentrations of FASL, TNF and IFNG were 100, 100 and 50 ng/ml, respectively. Cell viability was determined by optical density at 450 nm in a WST-1 assay. All values are expressed as a percentage of each control and represent mean ± SEM, n = 3 experiments. Different letters indicate significant differences (P < 0.05).
cells, FAS (Song et al., 2002) and DcR3 (Chen et al., 2009) expressions are both regulated by estradiol-17 and progesterone. Therefore, FAS and DcR3 expressions in the bovine endometrium may also be affected by steroid hormones.
Although FASL alone did not affect endometrial cell viability (Fig. 2), treatment or pretreatment with TNF + IFNG in combination with FASL significantly increased the sensitivity of the cells to FAS-mediated apoptosis (Fig. 2). TNF and IFNG produced by immune cells stimulate FAS expression
Fig. 3. Effects of TNF and IFNG on the expression of FAS mRNA in cultured bovine endometrial epithelial (A) and stromal (B) cells. These cells were exposed to TNF (100 ng/ml) and IFN (50 ng/ml) in the final 24 h of culture. All values are expressed as a percentage of each control and represent mean ± SEM, n = 4 experiments. Different letters indicate significant differences (P < 0.05).
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in various cell types (Yonehara et al., 1989; Taniguchi et al., 2002; Fluhr et al., 2007). In the present study, TNF + IFNG increased FAS mRNA expression in bovine endometrial cells (Fig. 3). In the bovine endometrium, the number of immunocytes increases and TNF expression peaks at the follicular stage (Cobb and Watson, 1995; Okuda et al., 2010; Oliveira et al., 2013). Therefore, the up-regulation of FAS expression by TNF and IFNG may help to induce apoptosis to remodel the endometrium at the follicular stage. Recently, matrix metalloproteinases (MMPs) have been reported to be expressed in bovine endometrium and to function in its remodeling (Mishra et al., 2010). MMPs have been suggested to have roles in the activation of FASL in the human endometrium, converting the membranebound form of FASL into the soluble form (Harada et al., 2004), so that the FAS/FASL-mediated apoptosis in the bovine endometrium may also be regulated by the action of MMPs. The mitochondrial apoptosis pathway is mainly regulated by BCL2 family members (Youle and Strasser, 2008). The BCL2 family has been suggested to play roles in the cyclic regulation of endometrial apoptosis because the BAX/BCL2 ratio has been correlated with endometrial apoptosis in several species other than cows (Tao et al., 1997; Vaskivuo et al., 2000; Mendoza-Rodriguez et al., 2003; Takagi-Morishita et al., 2003; Wei et al., 2005). We recently showed that the apoptosis of bovine endometrial cells peaked at the follicular and early luteal stages (Arai et al., 2013). In the present study, BCL2 expression was higher at the late luteal stage than at the early luteal and follicular stages, and the BAX/BCL2 ratio was higher at the early luteal stage than at the late luteal stage (Fig. 1E). These findings suggest that the mitochondrial apoptosis pathway, regulated by BCL2 family proteins, participates in endometrial apoptosis at the follicular and early luteal stages. On the other hand, BAX expression did not significantly change during the estrous cycle in the present study. Since BCL2 inhibits the activation of BAX, which induces permeabilization of the mitochondrial outer membrane (Youle and Strasser, 2008), apoptosis in the bovine endometrium may be depressed by the highly-expressed BCL2 during the late luteal stage. The signal from death receptors cleaves caspase-8, and cleaved caspase-8 activates the pro-apoptotic BH3-only protein, BID (Youle and Strasser, 2008). The activated BID not only inhibits the functions of pro-survival BCL2 family members by directly binding to them, but also activates BAX (Youle and Strasser, 2008). Hence, the BCL2 protein family as well as the BAX/BCL2 ratio are involved in regulating of the mitochondrial apoptotic pathway. In the present study, the BAX/BCL2 ratio was lower at the late luteal stage than at the early luteal stage, and the DcR3 protein expression was higher at the late luteal stage than at the early luteal and follicular stages. These results suggest that neither the intrinsic apoptotic pathways nor extrinsic apoptotic pathways are activated at the late luteal stage, resulting in less apoptosis in the endometrium. On the other hand, because the BAX/BCL2 ratio was high and the DcR3 protein expression was low at the follicular and early luteal stages, both apoptotic pathways are suggested to be activated to facilitate apoptosis at these stages.
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The overall results indicate that the expression of proand anti-apoptotic factors are regulated throughout the estrous cycle in the bovine endometrium, and suggest that both the extrinsic and intrinsic apoptosis pathways are related to the remodeling of the bovine endometrium. Our results also suggest that death ligands such as TNF, IFNG and FASL, produced by immune cells and the endometrium, regulate apoptosis resulting in dynamic functional and structural changes in bovine endometrium.
Conflict of interest statement None declared.
References Arai, M., Yoshioka, S., Tasaki, Y., Okuda, K., 2013. Remodeling of bovine endometrium throughout the estrous cycle. Anim. Reprod. Sci. 142, 1–9. Chen, H.F., Chen, J.S., Shun, C.T., Tsai, Y.F., Ho, H.N., 2009. Decoy receptor 3 expression during the menstrual cycle and pregnancy, and regulation by sex steroids in endometrial cells in vitro. Hum. Reprod. 24, 1350–1358. Cobb, S.P., Watson, E.D., 1995. Immunohistochemical study of immune cells in the bovine endometrium at different stages of the oestrous cycle. Res. Vet. Sci. 59, 238–241. Fluhr, H., Krenzer, S., Stein, G.M., Stork, B., Deperschmidt, M., Wallwiener, D., Wesselborg, S., Zygmunt, M., Licht, P., 2007. Interferon-␥ and tumor necrosis factor-␣ sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis. J. Cell. Sci. 120, 4126–4133. Fortier, M.A., Guilbault, L.A., Grasso, F., 1988. Specific properties of epithelial and stromal cells from the endometrium of cows. J. Reprod. Fertil. 83, 239–248. Harada, T., Kaponis, A., Iwabe, T., Taniguchi, F., Makrydimas, G., Sofikitis, N., Paschopoulos, M., Paraskevaidis, E., Terakawa, N., 2004. Apoptosis in human endometrium and endometriosis. Hum. Reprod. Update 10, 29–38. Hunt, J.S., Chen, H.L., Hu, X.L., Tabibzadeh, S., 1992. Tumor necrosis factor␣ messenger ribonucleic acid and protein in human endometrium. Biol. Reprod. 47, 141–147. Jin, Z., El-Deiry, W.S., 2005. Overview of cell death signaling pathways. Cancer Biol. Ther. 4, 139–163. Johnson, M.L., Redmer, D.A., Reynolds, L.P., 1997. Uterine growth, cell proliferation, and c-fos proto-oncogene expression throughout the estrous cycle in ewes. Biol. Reprod. 56, 393–401. Kerr, J.F., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257. Komiyama, J., Nishimura, R., Lee, H.Y., Sakumoto, R., Tetsuka, M., Acosta, T.J., Skarzynski, D.J., Okuda, K., 2008. Cortisol is a suppressor of apoptosis in bovine corpus luteum. Biol. Reprod. 78, 888–895. Liman, N., Alan, E., Bayram, G., Gurbulak, K., 2013. Expression of survivin, Bcl-2 and Bax proteins in the domestic cat (Felis catus) endometrium during the oestrus cycle. Reprod. Domest. Anim. 48, 33–45. Lin, W.W., Hsieh, S.L., 2011. Decoy receptor 3: a pleiotropic immunomodulator and biomarker for inflammatory diseases, autoimmune diseases and cancer. Biochem. Pharmacol. 81, 838–847. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2CT method. Methods 25, 402–408. Malayer, J.R., Woods, V.M., 1998. Expression of estrogen receptor and maintenance of hormone-responsive phenotype in bovine fetal uterine cells. Domest. Anim. Endocrinol. 15, 141–154. Mendoza-Rodriguez, C.A., Merchant-Larios, H., Segura-Valdez, M.L., Moreno-Mendoza, N., Cruz, M.E., Arteaga-Lopez, P., Camacho-Arroyo, I., Dominguez, R., Cerbon, M., 2003. c-fos and estrogen receptor gene expression pattern in the rat uterine epithelium during the estrous cycle. Mol. Reprod. Dev. 64, 379–388. Mishra, B., Kizaki, K., Koshi, K., Ushizawa, K., Takahashi, T., Hosoe, M., Sato, T., Ito, A., Hashizume, K., 2010. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle. Reprod. Biol. Endocrinol. 8, 60.
104
M. Arai et al. / Animal Reproduction Science 151 (2014) 97–104
Miyamoto, Y., Skarzynski, D.J., Okuda, K., 2000. Is tumor necrosis factor ␣ a trigger for the initiation of endometrial prostaglandin F2␣ release at luteolysis in cattle? Biol. Reprod. 62, 1109–1115. Murakami, S., Shibaya, M., Takeuchi, K., Skarzynski, D.J., Okuda, K., 2003. A passage and storage system for isolated bovine endometrial epithelial and stromal cells. J. Reprod. Dev. 49, 531–538. Nagata, S., 1997. Apoptosis by death factor. Cell 88, 355–365. Nagata, S., 1999. Fas ligand-induced apoptosis. Annu. Rev. Genet. 33, 29–55. Narkar, M., Kholkute, S., Nandedkar, T., 2006. Hormonal regulation of apoptosis in the endometrium of common marmosets (Callithrix jacchus). Theriogenology 66, 1194–1209. Okano, A., Ogawa, H., Takahashi, H., Geshi, M., 2007. Apoptosis in the porcine uterine endometrium during the estrous cycle, early pregnancy and post partum. J. Reprod. Dev. 53, 923–930. Okuda, K., Kito, S., Sumi, N., Sato, K., 1988. A study of the central cavity in the bovine corpus luteum. Vet. Rec. 123, 180–183. Okuda, K., Sakumoto, R., Okamoto, N., Acosta, T.J., Abe, H., Okada, H., Sinowatz, F., Skarzynski, D.J., 2010. Cellular localization of genes and proteins for tumor necrosis factor-␣ (TNF), TNF receptor types I and II in bovine endometrium. Mol. Cell. Endocrinol. 330, 41–48. Oliveira, L.J., Mansourri-Attia, N., Fahey, A.G., Browne, J., Forde, N., Roche, J.F., Lonergan, P., Fair, T., 2013. Characterization of the Th profile of the bovine endometrium during the oestrous cycle and early pregnancy. PLoS One 8, e75571. Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609–619. Osnes, T., Sandstad, O., Skar, V., Osnes, M., Kierulf, P., 1993. Total protein in common duct bile measured by acetonitrile precipitation and a micro bicinchoninic acid (BCA) method. Scand. J. Clin. Lab. Invest. 53, 757–763. Otsuki, Y., 2001. Apoptosis in human endometrium: apoptotic detection methods and signaling. Med. Electron Microsc. 34, 166–173. Philippeaux, M.M., Piguet, P.F., 1993. Expression of tumor necrosis factor␣ and its mRNA in the endometrial mucosa during the menstrual cycle. Am. J. Pathol. 143, 480–486. Pitti, R.M., Marsters, S.A., Lawrence, D.A., Roy, M., Kischkel, F.C., Dowd, P., Huang, A., Donahue, C.J., Sherwood, S.W., Baldwin, D.T., Godowski, P.J., Wood, W.I., Gurney, A.L., Hillan, K.J., Cohen, R.L., Goddard, A.D., Botstein, D., Ashkenazi, A., 1998. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 396, 699–703. Roberto da Costa, R.P., Serrao, P.M., Monteiro, S., Pessa, P., Silva, J.R., Ferreira-Dias, G., 2007. Caspase-3-mediated apoptosis and cell proliferation in the equine endometrium during the oestrous cycle. Reprod. Fertil. Dev. 19, 925–932. Sakumoto, R., Komatsu, T., Kasuya, E., Saito, T., Okuda, K., 2006. Expression of mRNAs for interleukin-4, interleukin-6 and their receptors in porcine corpus luteum during the estrous cycle. Domest. Anim. Endocrinol. 31, 246–257.
Song, J., Rutherford, T., Naftolin, F., Brown, S., Mor, G., 2002. Hormonal regulation of apoptosis and the Fas and Fas ligand system in human endometrial cells. Mol. Hum. Reprod. 8, 447–455. Strasser, A., O’Connor, L., Dixit, V.M., 2000. Apoptosis signaling. Annu. Rev. Biochem. 69, 217–245. Takagi-Morishita, Y., Yamada, N., Sugihara, A., Iwasaki, T., Tsujimura, T., Terada, N., 2003. Mouse uterine epithelial apoptosis is associated with expression of mitochondrial voltage-dependent anion channels, release of cytochrome C from mitochondria, and the ratio of Bax to Bcl-2 or Bcl-X. Biol. Reprod. 68, 1178–1184. Taniguchi, H., Yokomizo, Y., Okuda, K., 2002. Fas-Fas ligand system mediates luteal cell death in bovine corpus luteum. Biol. Reprod. 66, 754–759. Tanikawa, M., Lee, H.Y., Watanabe, K., Majewska, M., Skarzynski, D.J., Park, S.B., Lee, D.S., Park, C.K., Acosta, T.J., Okuda, K., 2008. Regulation of prostaglandin biosynthesis by interleukin-1 in cultured bovine endometrial cells. J. Endocrinol. 199, 425–434. Tao, X.J., Tilly, K.I., Maravei, D.V., Shifren, J.L., Krajewski, S., Reed, J.C., Tilly, J.L., Isaacson, K.B., 1997. Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax. J. Clin. Endocrinol. Metab. 82, 2738–2746. Van Herreweghe, F., Festjens, N., Declercq, W., Vandenabeele, P., 2010. Tumor necrosis factor-mediated cell death: to break or to burst, that’s the question. Cell. Mol. Life Sci. 67, 1567–1579. Vaskivuo, T.E., Stenback, F., Karhumaa, P., Risteli, J., Dunkel, L., Tapanainen, J.S., 2000. Apoptosis and apoptosis-related proteins in human endometrium. Mol. Cell. Endocrinol. 165, 75–83. Watanabe, H., Kanzaki, H., Narukawa, S., Inoue, T., Katsuragawa, H., Kaneko, Y., Mori, T., 1997. Bcl-2 and Fas expression in eutopic and ectopic human endometrium during the menstrual cycle in relation to endometrial cell apoptosis. Am. J. Obstet. Gynecol. 176, 360–368. Wei, P., Jin, X., Tao, S.X., Han, C.S., Liu, Y.X., 2005. Fas, FasL, Bcl-2, and Bax in the endometrium of rhesus monkey during the menstrual cycle. Mol. Reprod. Dev. 70, 478–484. Wood, G.A., Fata, J.E., Watson, K.L., Khokha, R., 2007. Circulating hormones and estrous stage predict cellular and stromal remodeling in murine uterus. Reproduction 133, 1035–1044. Yamashita, H., Otsuki, Y., Matsumoto, K., Ueki, K., Ueki, M., 1999. Fas ligand, Fas antigen and Bcl-2 expression in human endometrium during the menstrual cycle. Mol. Hum. Reprod. 5, 358–364. Yonehara, S., Ishii, A., Yonehara, M., 1989. A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J. Exp. Med. 169, 1747–1756. Youle, R.J., Strasser, A., 2008. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47–59.
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FAS/FASL-mediated cell death in the bovine endometrium夽 Miki Arai a , Shin Yoshioka a , Ryo Nishimura b , Kiyoshi Okuda a,∗ a Laboratory of Reproductive Physiology, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan b Laboratory of Theriogenology, Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8550, Japan
a r t i c l e
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Article history: Received 4 June 2014 Received in revised form 26 September 2014 Accepted 5 October 2014 Available online 14 October 2014 Keywords: Cattle Endometrium Remodeling Apoptosis
a b s t r a c t In many mammals, endometrial cells are remodeled by apoptosis and cell proliferation throughout the estrous cycle. Although apoptosis is known to be induced by various factors involving two major apoptotic pathways (the death receptor- and mitochondria-mediated pathways), how it is regulated in the bovine endometrium is unclear. We examined (1) the cyclic expressions of apoptosis-related factors, FAS, DcR3, BCL2 and BAX, in the bovine endometrium and (2) the effect of death ligands on the viability of, and FAS mRNA expression in, cultured bovine endometrial epithelial and stromal cells. FAS expression did not change during the estrous cycle, whereas DcR3 expression was higher at the mid and late luteal stages than at the early luteal and follicular stages. BCL2 expression was higher at the late luteal stage than at the early luteal and follicular stages, and the BAX/BCL2 ratio was higher at the early luteal stage than at the late luteal stage. Treatment or pretreatment with tumor necrosis factor-␣ (TNF) + interferon ␥ (IFNG) in combination with FAS ligand significantly reduced the viability of both epithelial and stromal cells. Furthermore, TNF + IFNG treatment significantly increased the expression of FAS mRNA in both types of endometrial cells. The overall results suggest that both extrinsic and intrinsic pathways are involved in remodeling the bovine endometrium throughout the estrous cycle, and that the death ligands produced by immune cells and the endometrium play important roles in inducing cyclic endometrial cell death. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In many mammals, endometrium changes morphologically and functionally during the estrous cycle, and is remodeled by cyclic cell proliferation and apoptosis (Johnson et al., 1997; Okano et al., 2007; Roberto da Costa et al., 2007; Wood et al., 2007). Recently, we demonstrated that both cell proliferation and apoptosis (programmed cell
夽 This research was supported by a Grant-in-Aid for Scientific Research (No. 24380155) of the Japan Society for the Promotion of Science (JSPS). ∗ Corresponding author. Tel.: +81 86 251 833; fax: +81 86 251 8349. E-mail address: [email protected] (K. Okuda). http://dx.doi.org/10.1016/j.anireprosci.2014.10.004 0378-4320/© 2014 Elsevier B.V. All rights reserved.
death) are active in the bovine endometrium at the follicular and early luteal stages (Arai et al., 2013). Apoptosis has critical roles in the development and functional maintenance of a variety of tissues including the human endometrium (Kerr et al., 1972; Strasser et al., 2000; Harada et al., 2004; Jin and El-Deiry, 2005). Apoptotic signaling occurs through two major pathways, the death receptor-mediated (extrinsic) pathway and the mitochondria-mediated (intrinsic) pathway (Nagata, 1997; Jin and El-Deiry, 2005). The extrinsic apoptosis pathway is mainly regulated by ligands and receptors of the tumor necrosis factor superfamily, which are referred to as “death ligands” and “death receptors” (Strasser et al., 2000; Jin and El-Deiry, 2005). Tumor necrosis factor-␣ (TNF) is a cytokine
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that participates in various physiological and inflammatory responses by binding to TNF receptor (TNFR) 1 and TNFR2 at the cell membrane (Nagata, 1997). TNFR1 has a cytoplasmic death domain and induces not only apoptosis, but also the activation of NF-B (Nagata, 1997). In contrast, TNFR2 lacks a death domain, and is suggested to modulate the actions of TNFR1 (Van Herreweghe et al., 2010). FAS, a TNF superfamily receptor, is known to induce apoptotic cell death when it binds to FAS ligand (FASL) (Jin and El-Deiry, 2005), which is mainly expressed in activated T lymphocytes and natural killer (NK) cells (Nagata, 1999). The binding of ligands to TNFR1 or FAS recruits and activates a series of cysteine proteases, called caspases (Jin and El-Deiry, 2005). The binding induces caspase-8 activation through the adaptor protein Fas-associated death domain and then induces apoptosis by means of activation of caspase-3 (Jin and El-Deiry, 2005). FASL binds not only to FAS but also to decoy receptor 3 (DcR3), which is a soluble decoy receptor that lacks a transmembrane domain and that neutralizes FAS-induced apoptosis (Pitti et al., 1998; Lin and Hsieh, 2011). The mitochondrial pathway is mainly regulated by BCL2 family proteins (Youle and Strasser, 2008). BCL2 family members are categorized into three groups based on their functions and structures: (1) pro-survival BCL2 family members (e.g., BCL2, BCL-XL), (2) pro-apoptotic BCL2 family members (e.g., BAX, BAK) and (3) proapoptotic BH3-only proteins (e.g., BID, BAD, BIM) (Youle and Strasser, 2008). The pro-apoptotic BAX permeabilizes the outer mitochondrial membrane and releases cytochrome c (Youle and Strasser, 2008). Cytochrome c released into the cytosol binds to apoptotic proteaseactivating factor-1 (APAF1) and then activates caspase-9 to turn on caspase-3 (Youle and Strasser, 2008). By contrast, the anti-apoptotic BCL2 inhibits BAX activation and the subsequent cytochrome c release (Oltvai et al., 1993; Jin and El-Deiry, 2005). Therefore, the ratio of BAX to BCL2 is thought to be important for decision of cellular survival (Oltvai et al., 1993). Activation of caspase8 in the death receptor-mediated pathway activates a pro-apoptotic BH3-only protein, BID, which promotes apoptosis via the mitochondrial pathway (Youle and Strasser, 2008). Some members of TNF and TNFR superfamilies and BCL2 family are expressed in the endometrium throughout the ovarian cycle in cattle (Okuda et al., 2010), cat (Liman et al., 2013), monkey (Wei et al., 2005), marmoset (Narkar et al., 2006) and human (Hunt et al., 1992; Philippeaux and Piguet, 1993; Watanabe et al., 1997; Yamashita et al., 1999; Tao et al., 1997; Vaskivuo et al., 2000). These include TNF (Hunt et al., 1992; Philippeaux and Piguet, 1993), FAS (Watanabe et al., 1997; Yamashita et al., 1999; Wei et al., 2005), DcR3 (Chen et al., 2009) and BCL2 and BAX (Tao et al., 1997; Vaskivuo et al., 2000; Wei et al., 2005; Narkar et al., 2006; Liman et al., 2013). In many of those species, cyclic endometrial apoptosis is controlled by periodic expressions of apoptosis regulatory factors (Harada et al., 2004). In the endometrium of human and other primates, BAX expressions are high during the secretory (luteal) phase (Tao et al., 1997; Wei et al., 2005; Narkar et al., 2006), while the changes in BCL2 expression during the menstrual cycle
are not consistent among reports (Watanabe et al., 1997; Tao et al., 1997; Yamashita et al., 1999; Narkar et al., 2006). Serum DcR3 levels are high during the mid luteal phase and during normal pregnancy in the human endometrium (Chen et al., 2009), while FAS protein expression does not change throughout the menstrual cycle (Watanabe et al., 1997; Yamashita et al., 1999). We recently reported that the bovine endometrium is also remodeled throughout the estrous cycle (Arai et al., 2013). In the late luteal and the follicular stages in the bovine endometrium, increases are observed in TNF mRNA levels (Okuda et al., 2010) and in the numbers of T lymphocytes and NK cells, both of which produce FASL, TNF and interferon ␥ (IFNG) (Cobb and Watson, 1995; Oliveira et al., 2013). However, it is unclear how the expressions of other pro- and anti-apoptotic factors in the bovine endometrium during the estrous cycle are involved in regulating endometrial apoptosis. To understand how apoptosis is regulated during remodeling of bovine endometrium, we examined (1) cyclic expressions of FAS, DcR3, BCL2 and BAX in the bovine endometrium and (2) the effects of death ligands (TNF, IFNG and FASL), on the viability of, and FAS mRNA expression in, cultured bovine endometrial epithelial and stromal cells. 2. Materials and methods 2.1. Collection of endometrial tissues Apparently healthy uteri of Holstein cows without a visible conceptus were obtained within 10–20 min after exsanguinations and were immediately transported to the laboratory. Stages of the estrous cycle were confirmed by macroscopic observation of the ovary and uterus as described previously (Okuda et al., 1988; Miyamoto et al., 2000). For protein determination, endometrial tissues (n = 4 cows/stages) were collected from cows at four different stages of the estrous cycle (estrus = Day 0; early luteal: Days 2–3; mid luteal: Days 8–12; late luteal: Days 15–17 and follicular stage: Days 19–21). Intercaruncular endometrial tissues from the uterine horn, ipsilateral to the CL or the dominant follicle, were used for experiments. Endometrial tissues were immediately frozen in liquid nitrogen, and stored at −80 ◦ C until processed for protein isolation. For cell culture experiments, the uterus was submerged in ice-cold physiological saline and transported to the laboratory within 1–1.5 h on ice. 2.2. Isolation and culture of endometrial cells Uteri at Days 2–5 after ovulation were utilized for cell culture. Epithelial and stromal cells from bovine endometrium were separated as described previously (Murakami et al., 2003; Arai et al., 2013). After the collecting epithelial cells by incubating with trypsin [0.3% (w/v) (>7500 BAEE units/mg solid; T9201; Sigma-Aldrich, St. Louis, MO, USA)], stromal cells were isolated by dissociation of minced endometrial tissues by incubating with collagenase [0.05% (w/v); no. CLS1; Worthington Biochemical Co., Lakewood, NJ, USA] and DNase I [0.005% (w/v); DNP2; BBI Enzymes, Cardiff, UK]. The isolated epithelial
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and stromal cells were counted using a hemocytometer, and the viability of both cell types was greater than 85% as assessed by 0.5% (w/v) trypan blue dye exclusion. Final pellets of the epithelial and stromal cells were separately resuspended in culture medium (DMEM/F-12, 1:1 Mixture: 12400-024; Life Technologies, Carlsbad, CA, USA) supplemented with 10% (v/v) bovine serum (16170078; Life Technologies), 20 g/ml gentamicin (G1397; Sigma-Aldrich) and 2 mg/ml amphotericin B (A9528; Sigma-Aldrich). The homogeneity of stromal cells and epithelial cells was evaluated using immunofluorescent staining for specific markers of epithelial (cytokeratin) and stromal cells (vimentin) as described previously (Malayer and Woods, 1998; Tanikawa et al., 2008). The epithelial cell contamination of stromal cells was about 1%, and the stromal cells contamination of epithelial cells was <1%. Stromal cells were seeded at a density of 1.0 × 105 viable cells/ml in 96-well cluster dishes (3860-096; Asahi Techno Glass, Chiba, Japan) for cell-viability experiments or 24-well cluster dishes (no. 662160; Greiner Bio-One, Frickenhausen, Germany) for FAS mRNA expression determinations. To purify the stromal preparation, the medium was changed 2 h after plating, by which time selective attachment of stromal cells had occurred (Fortier et al., 1988). Epithelial cells were seeded at a density of 1.0 × 105 viable cells/ml in 75 cm2 culture flasks (no. 658175; Greiner Bio-One) for trypsinization. Since epithelial cells attached 24–48 h after plating, the medium in the epithelial cell culture was replaced 48 h after plating. When epithelial cells reached sub-confluence, the cells were trypsinized to purify the epithelial cells. Briefly, epithelial cells in the culture flask were washed with phosphate-buffered saline (PBS) twice. After the washing, 0.02% (w/v) porcine trypsin (1000–2000 BAEE units/mg solid; Sigma-Aldrich) with 0.008% (w/v) EDTA (SigmaAldrich) in PBS was added to the flask and the cells were incubated for 5 min at 38 ◦ C to detach stromal cells. Then, the solution containing the stromal cells was removed, and the remaining cells were incubated with 2 mmol/l EDTA in PBS for 2 min at 38 ◦ C. After the incubation, the solution was removed and the cells were washed with PBS, and the cells were incubated for 10 min at 38 ◦ C with 0.02% (w/v) bovine trypsin in PBS. After the incubation, the cells were washed by centrifugation (180 × g for 10 min at 4 ◦ C) with culture medium. Then, epithelial cells were seeded at the same density (1.0 × 105 viable cells/ml) as stromal cells, and cultured at 38 ◦ C in a humidified atmosphere of 5% CO2 in air until the cells reached confluence. 2.3. Experiment 1: FAS, DcR3, BCL2 and BAX protein expressions throughout the estrous cycle FAS, DcR3, BCL2 and BAX protein levels in endometrial tissues were assessed by Western blot analysis (n = 4 experiments). Endometrial tissues were homogenized on ice in the homogenization buffer by a tissue homogenizer (NS-50; Physcotron; Niti-on, Chiba, Japan), followed by filtration with a metal wire mesh (150 m). For each protein analysis, tissue homogenates were centrifuged at 600 × g for 10 min and these supernatants were
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recentrifuged at 9000 × g for 30 min. After centrifugation, supernatant were collected as cytoplasmic fractionations for FAS and DcR3 protein analysis and precipitates were collected as mitochondrial fractionations for BCL2 and BAX protein analysis. Protein concentrations were determined by BCA method (Osnes et al., 1993). The proteins were heated with SDS gel-loading buffer containing 1% (v/v) -mercaptoethanol (137-06862; Wako Pure Chemical Industries) at 95 ◦ C for 10 min. Samples (25 g protein) were subjected to SDS-PAGE (15%) for 1.5 h at 200 V. Separated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (RPN303F; GE Healthcare, Milwaukee, WI, USA) for 30 min at 200 mA using a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA). The membrane was washed in TBS with 0.1% Tween 20 (TBST), incubated in PVDF Blocking Reagent for Can Get Signal (NYPBR01; TOYOBO, Osaka, Japan) for 60 min at room temperature, incubated separately with specific primary antibodies to FAS (55 kDa: 05-351; Merck KGaA, Darmstadt, Germany; 1:500 dilution), DcR3 (32 kDa: no. 4758; Cell Signaling Technology, Boston, MA, USA; 1:1000 dilution), BCL2 (28 kDa: no. 2876; Cell Signaling Technology; 1:1000 dilution), BAX (20 kDa: no. 2772; Cell Signaling Technology; 1:1000 dilution) and ACTB (42 kDa: A2228; Sigma-Aldrich; 1:4000 dilution) in Can Get Signal Immunoreaction Enhancer Solution 1 (NKB201; TOYOBO) overnight at 4 ◦ C, incubated with secondary antibodies to FAS (554017; HRP Goat Anti-rat Ig; Becton, Dickinson and Company, Franklin Lakes, NJ, USA; 1:500 dilution), to DcR3, BCL2 and BAX (NA934; antirabbit Ig, HRP-linked whole antibody produced in donkey; GE Healthcare; 1:4000 dilution) and to ACTB (NA931; anti-mouse Ig, HRP-linked whole antibody produced in sheep; GE Healthcare; 1:40,000 dilution) in Can Get Signal Immunoreaction Enhancer Solution 2 (NKB-301; TOYOBO) for 60 min at room temperature, incubated in Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500; Merck) and captured using a ChemiDocTM XRS+ System (Bio-Rad). The intensity of the immunological reaction was estimated by measuring the optical density in the defined area by computerized densitometry using an Image LabTM software (170-8265J1PC; Bio-Rad). 2.4. Experiment 2: Effect of FASL, TNF and IFNG on cell viability When cells were confluent, the culture medium was replaced with DMEM/F-12 without phenol red medium-BSA [DMEM/F-12 without phenol red (D2906; Sigma-Aldrich) supplemented with 0.1% (w/v) BSA, 5 ng/ml sodium selenite (S5261; Sigma-Aldrich), 0.5 mM ascorbic acid (031-12061; Wako Pure Chemical Industries, Ltd., Osaka, Japan), 5 mg/ml transferrin (T4132; SigmaAldrich) and 20 mg/ml gentamicin]. Then, the cells were cultured in the presence or absence of stimulants [TNF (100 ng/ml, HF-13; kindly donated by Dainippon pharmaceutical, Osaka, Japan), IFNG (50 ng/ml, kindly donated by Dr. Inumaru, National Institute of Animal Health, Ibaraki, Japan) and FASL (50 ng/ml, F0427; SigmaAldrich)] for 24 h, after pretreatment with TNF and IFNG for 24 h (n = 3 experiments) or no pretreatment
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(n = 3 experiments). Cell viability was determined by a Dojindo Cell Counting Kit, including WST-1 (no. 34506463; Dojindo, Kumamoto, Japan) as described previously (Komiyama et al., 2008). Briefly, WST-1 [2-(4-iodophenyl)3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H-tetrazolium, monosodium salt], a derivative of MTT (3-[4,5-dimethyl2thiazolyl]-2,5-diphenyl-2H-tetrazolium/Br), is a tetrazolium salt reduced to formazan dye by live cells containing active mitochondria. For the viability assay, the culture medium was replaced with 100 ml DMEM/F-12 medium without phenol red, and then 10 ml of assay solution [0.3% WST-1, 0.2 mM 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS) in PBS, pH 7.4] was added to each well. The cells were then incubated for 3 h at 38 ◦ C. The absorbance (A) was read at 450 nm using a microplate reader (model 450; Bio-Rad). Cell viability (%) was calculated as cell viability (%) = 100 × (Atest /Acontrol ), where Acontrol is the mean A of non-treated wells, and Atest is the mean A of FASL-, TNF + IFNG-, and FASL + TNF + IFNGtreated wells. The mean A of wells in the absence of the cells was subtracted from the mean A of all experimental wells.
2.5. Experiment 3: Effect of TNF and IFNG on FAS mRNA expression When cells were confluent, the culture medium was replaced with DMEM/F-12 without phenol red mediumBSA in the presence or absence of TNF (100 ng/ml) and IFNG (50 ng/ml) for 24 h (n = 4 experiments). After 24 h of culture, total RNA was extracted from endometrial cells using TRIsure (no. BIO-38033; Bioline, London, UK) according to the manufacturer’s directions. Total RNA concentration (g/ml) was calculated as 40 × A260 . The purity of RNA was evaluated by the ratio of A260 /A280 , and samples with ratio 2.0
2.6. Statistical analysis All experimental data are shown as the mean ± SEM. The statistical significance of differences was assessed by analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test using GraphPad Prism ver. 6.0 for Mac (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Experiment 1: FAS, DcR3, BCL2 and BAX protein expression throughout the estrous cycle FAS and DcR3 were expressed in the bovine endometrium throughout the estrous cycle (Fig. 1A and B). FAS expression in the bovine endometrium did not significantly change during the estrous cycle (Fig. 1A), whereas DcR3 expression was higher at the mid and late luteal stages than at the early luteal and follicular stages (Fig. 1B; P < 0.05). BCL2 and BAX protein were also expressed in the bovine endometrium throughout the estrous cycle (Fig. 1C and D). BCL2 expression was lower at the early luteal and follicular stages than at the late luteal stage (Fig. 1C; P < 0.05). BAX expression did not significantly change during the estrous cycle (Fig. 1D). Furthermore, the BAX/BCL2 ratio was higher at the early luteal stage than at the late luteal stage (Fig. 1E; P < 0.05). 3.2. Experiment 2: Effect of FASL and TNF + IFNG on cell viability FASL alone did not affect the viability in epithelial and stromal cells (Fig. 2). Furthermore, TNF + IFNG with or without FASL did not reduce the viability of epithelial cells (Fig. 2A). On the other hand, the viability of stromal cells was decreased to approximately 60% in cultures with FASL in combination with TNF + IFNG (Fig. 2B; P < 0.05). After the pretreatment with TNF + IFNG for 24 h, FASL or TNF + IFNG alone reduced the viability of epithelial cells (Fig. 2C; P < 0.05). TNF + IFNG in combination with FASL reduced the viability of epithelial cells compared with FASL or TNF + IFNG alone (Fig. 2C; P < 0.05). In stromal cells after the pretreatment with TNF + IFNG, TNF + IFNG decreased cell viability (Fig. 2D; P < 0.05). Moreover, FASL with or without TNF + IFNG significantly reduced the viability of stromal cells to less than 30% (Fig. 2D; P < 0.05). 3.3. Experiment 3: Effect of TNF + IFNG on FAS mRNA expression TNF + IFNG significantly increased the expression of FAS mRNA in epithelial and stromal cells (Fig. 3; P < 0.05). 4. Discussion In bovine endometrium, it is unclear whether apoptosis is related to the regulation of its physiological function. Recently, we obtained evidence for apoptosis in bovine endometrium by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase-3
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Fig. 1. Changes in FAS (A), DcR3 (B), BCL2 (C) and BAX (D) protein expressions (relative ratios to ACTB protein expression) and BAX/BCL2 ratio (E) in the bovine endometrium throughout the estrous cycle (mean ± SEM, n = 4 experiments). Different letters indicate significant differences (P < 0.05).
(CCP3) (Arai et al., 2013). We also found that the population of apoptotic endometrial cells changed during the estrous cycle (Arai et al., 2013). In the present study, we confirmed that FAS, one of death receptors, is expressed in bovine endometrium. These findings indicate that apoptosis is an important regulatory mechanism for bovine endometrial function. In addition, studies with cultured endometrial cells showed that cell death was induced by FASL in the presence of TNF and IFNG. This result suggests that FAS/FASL-mediated cell death is related to the cyclic changes in bovine endometrium. FAS-mediated apoptosis participates in the regulation of human endometrial function (Otsuki, 2001; Song et al., 2002; Harada et al., 2004). DcR3, a member of the TNFR superfamily, competes with FAS for binding to FASL and inhibits FASL-induced apoptosis (Pitti et al., 1998), so that it
is necessary to determine the expressions of both FAS and DcR3 to evaluate FASL-induced apoptosis. In the present study, FAS expression did not significantly change during the estrous cycle (Fig. 1A), while DcR3 expression was higher at the mid and late luteal stages than at the early luteal and follicular stages in the bovine endometrium (Fig. 1B). These findings agreed with our previous findings that cleaved caspase-3 expression was higher at the early luteal and follicular stages than at the mid and the late luteal stages in the bovine endometrium (Arai et al., 2013). The number of T lymphocytes and NK cells, which are known to produce FASL, increases in bovine uterus during luteolysis (Cobb and Watson, 1995; Oliveira et al., 2013). The increase of those cells and the decrease of DcR3 expression may induce apoptosis in the bovine endometrium during luteolysis. In human endometrial
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Fig. 2. Effects of FASL and TNF + IFNG on the viability of cultured bovine endometrial cells. (A and C): Epithelial cells without and with 24-h TNF + IFNG pretreatment, respectively. (B and D): Stromal cells without and with 24-h TNF + IFNG pretreatment, respectively. Concentrations of FASL, TNF and IFNG were 100, 100 and 50 ng/ml, respectively. Cell viability was determined by optical density at 450 nm in a WST-1 assay. All values are expressed as a percentage of each control and represent mean ± SEM, n = 3 experiments. Different letters indicate significant differences (P < 0.05).
cells, FAS (Song et al., 2002) and DcR3 (Chen et al., 2009) expressions are both regulated by estradiol-17 and progesterone. Therefore, FAS and DcR3 expressions in the bovine endometrium may also be affected by steroid hormones.
Although FASL alone did not affect endometrial cell viability (Fig. 2), treatment or pretreatment with TNF + IFNG in combination with FASL significantly increased the sensitivity of the cells to FAS-mediated apoptosis (Fig. 2). TNF and IFNG produced by immune cells stimulate FAS expression
Fig. 3. Effects of TNF and IFNG on the expression of FAS mRNA in cultured bovine endometrial epithelial (A) and stromal (B) cells. These cells were exposed to TNF (100 ng/ml) and IFN (50 ng/ml) in the final 24 h of culture. All values are expressed as a percentage of each control and represent mean ± SEM, n = 4 experiments. Different letters indicate significant differences (P < 0.05).
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in various cell types (Yonehara et al., 1989; Taniguchi et al., 2002; Fluhr et al., 2007). In the present study, TNF + IFNG increased FAS mRNA expression in bovine endometrial cells (Fig. 3). In the bovine endometrium, the number of immunocytes increases and TNF expression peaks at the follicular stage (Cobb and Watson, 1995; Okuda et al., 2010; Oliveira et al., 2013). Therefore, the up-regulation of FAS expression by TNF and IFNG may help to induce apoptosis to remodel the endometrium at the follicular stage. Recently, matrix metalloproteinases (MMPs) have been reported to be expressed in bovine endometrium and to function in its remodeling (Mishra et al., 2010). MMPs have been suggested to have roles in the activation of FASL in the human endometrium, converting the membranebound form of FASL into the soluble form (Harada et al., 2004), so that the FAS/FASL-mediated apoptosis in the bovine endometrium may also be regulated by the action of MMPs. The mitochondrial apoptosis pathway is mainly regulated by BCL2 family members (Youle and Strasser, 2008). The BCL2 family has been suggested to play roles in the cyclic regulation of endometrial apoptosis because the BAX/BCL2 ratio has been correlated with endometrial apoptosis in several species other than cows (Tao et al., 1997; Vaskivuo et al., 2000; Mendoza-Rodriguez et al., 2003; Takagi-Morishita et al., 2003; Wei et al., 2005). We recently showed that the apoptosis of bovine endometrial cells peaked at the follicular and early luteal stages (Arai et al., 2013). In the present study, BCL2 expression was higher at the late luteal stage than at the early luteal and follicular stages, and the BAX/BCL2 ratio was higher at the early luteal stage than at the late luteal stage (Fig. 1E). These findings suggest that the mitochondrial apoptosis pathway, regulated by BCL2 family proteins, participates in endometrial apoptosis at the follicular and early luteal stages. On the other hand, BAX expression did not significantly change during the estrous cycle in the present study. Since BCL2 inhibits the activation of BAX, which induces permeabilization of the mitochondrial outer membrane (Youle and Strasser, 2008), apoptosis in the bovine endometrium may be depressed by the highly-expressed BCL2 during the late luteal stage. The signal from death receptors cleaves caspase-8, and cleaved caspase-8 activates the pro-apoptotic BH3-only protein, BID (Youle and Strasser, 2008). The activated BID not only inhibits the functions of pro-survival BCL2 family members by directly binding to them, but also activates BAX (Youle and Strasser, 2008). Hence, the BCL2 protein family as well as the BAX/BCL2 ratio are involved in regulating of the mitochondrial apoptotic pathway. In the present study, the BAX/BCL2 ratio was lower at the late luteal stage than at the early luteal stage, and the DcR3 protein expression was higher at the late luteal stage than at the early luteal and follicular stages. These results suggest that neither the intrinsic apoptotic pathways nor extrinsic apoptotic pathways are activated at the late luteal stage, resulting in less apoptosis in the endometrium. On the other hand, because the BAX/BCL2 ratio was high and the DcR3 protein expression was low at the follicular and early luteal stages, both apoptotic pathways are suggested to be activated to facilitate apoptosis at these stages.
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The overall results indicate that the expression of proand anti-apoptotic factors are regulated throughout the estrous cycle in the bovine endometrium, and suggest that both the extrinsic and intrinsic apoptosis pathways are related to the remodeling of the bovine endometrium. Our results also suggest that death ligands such as TNF, IFNG and FASL, produced by immune cells and the endometrium, regulate apoptosis resulting in dynamic functional and structural changes in bovine endometrium.
Conflict of interest statement None declared.
References Arai, M., Yoshioka, S., Tasaki, Y., Okuda, K., 2013. Remodeling of bovine endometrium throughout the estrous cycle. Anim. Reprod. Sci. 142, 1–9. Chen, H.F., Chen, J.S., Shun, C.T., Tsai, Y.F., Ho, H.N., 2009. Decoy receptor 3 expression during the menstrual cycle and pregnancy, and regulation by sex steroids in endometrial cells in vitro. Hum. Reprod. 24, 1350–1358. Cobb, S.P., Watson, E.D., 1995. Immunohistochemical study of immune cells in the bovine endometrium at different stages of the oestrous cycle. Res. Vet. Sci. 59, 238–241. Fluhr, H., Krenzer, S., Stein, G.M., Stork, B., Deperschmidt, M., Wallwiener, D., Wesselborg, S., Zygmunt, M., Licht, P., 2007. Interferon-␥ and tumor necrosis factor-␣ sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis. J. Cell. Sci. 120, 4126–4133. Fortier, M.A., Guilbault, L.A., Grasso, F., 1988. Specific properties of epithelial and stromal cells from the endometrium of cows. J. Reprod. Fertil. 83, 239–248. Harada, T., Kaponis, A., Iwabe, T., Taniguchi, F., Makrydimas, G., Sofikitis, N., Paschopoulos, M., Paraskevaidis, E., Terakawa, N., 2004. Apoptosis in human endometrium and endometriosis. Hum. Reprod. Update 10, 29–38. Hunt, J.S., Chen, H.L., Hu, X.L., Tabibzadeh, S., 1992. Tumor necrosis factor␣ messenger ribonucleic acid and protein in human endometrium. Biol. Reprod. 47, 141–147. Jin, Z., El-Deiry, W.S., 2005. Overview of cell death signaling pathways. Cancer Biol. Ther. 4, 139–163. Johnson, M.L., Redmer, D.A., Reynolds, L.P., 1997. Uterine growth, cell proliferation, and c-fos proto-oncogene expression throughout the estrous cycle in ewes. Biol. Reprod. 56, 393–401. Kerr, J.F., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257. Komiyama, J., Nishimura, R., Lee, H.Y., Sakumoto, R., Tetsuka, M., Acosta, T.J., Skarzynski, D.J., Okuda, K., 2008. Cortisol is a suppressor of apoptosis in bovine corpus luteum. Biol. Reprod. 78, 888–895. Liman, N., Alan, E., Bayram, G., Gurbulak, K., 2013. Expression of survivin, Bcl-2 and Bax proteins in the domestic cat (Felis catus) endometrium during the oestrus cycle. Reprod. Domest. Anim. 48, 33–45. Lin, W.W., Hsieh, S.L., 2011. Decoy receptor 3: a pleiotropic immunomodulator and biomarker for inflammatory diseases, autoimmune diseases and cancer. Biochem. Pharmacol. 81, 838–847. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2CT method. Methods 25, 402–408. Malayer, J.R., Woods, V.M., 1998. Expression of estrogen receptor and maintenance of hormone-responsive phenotype in bovine fetal uterine cells. Domest. Anim. Endocrinol. 15, 141–154. Mendoza-Rodriguez, C.A., Merchant-Larios, H., Segura-Valdez, M.L., Moreno-Mendoza, N., Cruz, M.E., Arteaga-Lopez, P., Camacho-Arroyo, I., Dominguez, R., Cerbon, M., 2003. c-fos and estrogen receptor gene expression pattern in the rat uterine epithelium during the estrous cycle. Mol. Reprod. Dev. 64, 379–388. Mishra, B., Kizaki, K., Koshi, K., Ushizawa, K., Takahashi, T., Hosoe, M., Sato, T., Ito, A., Hashizume, K., 2010. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle. Reprod. Biol. Endocrinol. 8, 60.
104
M. Arai et al. / Animal Reproduction Science 151 (2014) 97–104
Miyamoto, Y., Skarzynski, D.J., Okuda, K., 2000. Is tumor necrosis factor ␣ a trigger for the initiation of endometrial prostaglandin F2␣ release at luteolysis in cattle? Biol. Reprod. 62, 1109–1115. Murakami, S., Shibaya, M., Takeuchi, K., Skarzynski, D.J., Okuda, K., 2003. A passage and storage system for isolated bovine endometrial epithelial and stromal cells. J. Reprod. Dev. 49, 531–538. Nagata, S., 1997. Apoptosis by death factor. Cell 88, 355–365. Nagata, S., 1999. Fas ligand-induced apoptosis. Annu. Rev. Genet. 33, 29–55. Narkar, M., Kholkute, S., Nandedkar, T., 2006. Hormonal regulation of apoptosis in the endometrium of common marmosets (Callithrix jacchus). Theriogenology 66, 1194–1209. Okano, A., Ogawa, H., Takahashi, H., Geshi, M., 2007. Apoptosis in the porcine uterine endometrium during the estrous cycle, early pregnancy and post partum. J. Reprod. Dev. 53, 923–930. Okuda, K., Kito, S., Sumi, N., Sato, K., 1988. A study of the central cavity in the bovine corpus luteum. Vet. Rec. 123, 180–183. Okuda, K., Sakumoto, R., Okamoto, N., Acosta, T.J., Abe, H., Okada, H., Sinowatz, F., Skarzynski, D.J., 2010. Cellular localization of genes and proteins for tumor necrosis factor-␣ (TNF), TNF receptor types I and II in bovine endometrium. Mol. Cell. Endocrinol. 330, 41–48. Oliveira, L.J., Mansourri-Attia, N., Fahey, A.G., Browne, J., Forde, N., Roche, J.F., Lonergan, P., Fair, T., 2013. Characterization of the Th profile of the bovine endometrium during the oestrous cycle and early pregnancy. PLoS One 8, e75571. Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609–619. Osnes, T., Sandstad, O., Skar, V., Osnes, M., Kierulf, P., 1993. Total protein in common duct bile measured by acetonitrile precipitation and a micro bicinchoninic acid (BCA) method. Scand. J. Clin. Lab. Invest. 53, 757–763. Otsuki, Y., 2001. Apoptosis in human endometrium: apoptotic detection methods and signaling. Med. Electron Microsc. 34, 166–173. Philippeaux, M.M., Piguet, P.F., 1993. Expression of tumor necrosis factor␣ and its mRNA in the endometrial mucosa during the menstrual cycle. Am. J. Pathol. 143, 480–486. Pitti, R.M., Marsters, S.A., Lawrence, D.A., Roy, M., Kischkel, F.C., Dowd, P., Huang, A., Donahue, C.J., Sherwood, S.W., Baldwin, D.T., Godowski, P.J., Wood, W.I., Gurney, A.L., Hillan, K.J., Cohen, R.L., Goddard, A.D., Botstein, D., Ashkenazi, A., 1998. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 396, 699–703. Roberto da Costa, R.P., Serrao, P.M., Monteiro, S., Pessa, P., Silva, J.R., Ferreira-Dias, G., 2007. Caspase-3-mediated apoptosis and cell proliferation in the equine endometrium during the oestrous cycle. Reprod. Fertil. Dev. 19, 925–932. Sakumoto, R., Komatsu, T., Kasuya, E., Saito, T., Okuda, K., 2006. Expression of mRNAs for interleukin-4, interleukin-6 and their receptors in porcine corpus luteum during the estrous cycle. Domest. Anim. Endocrinol. 31, 246–257.
Song, J., Rutherford, T., Naftolin, F., Brown, S., Mor, G., 2002. Hormonal regulation of apoptosis and the Fas and Fas ligand system in human endometrial cells. Mol. Hum. Reprod. 8, 447–455. Strasser, A., O’Connor, L., Dixit, V.M., 2000. Apoptosis signaling. Annu. Rev. Biochem. 69, 217–245. Takagi-Morishita, Y., Yamada, N., Sugihara, A., Iwasaki, T., Tsujimura, T., Terada, N., 2003. Mouse uterine epithelial apoptosis is associated with expression of mitochondrial voltage-dependent anion channels, release of cytochrome C from mitochondria, and the ratio of Bax to Bcl-2 or Bcl-X. Biol. Reprod. 68, 1178–1184. Taniguchi, H., Yokomizo, Y., Okuda, K., 2002. Fas-Fas ligand system mediates luteal cell death in bovine corpus luteum. Biol. Reprod. 66, 754–759. Tanikawa, M., Lee, H.Y., Watanabe, K., Majewska, M., Skarzynski, D.J., Park, S.B., Lee, D.S., Park, C.K., Acosta, T.J., Okuda, K., 2008. Regulation of prostaglandin biosynthesis by interleukin-1 in cultured bovine endometrial cells. J. Endocrinol. 199, 425–434. Tao, X.J., Tilly, K.I., Maravei, D.V., Shifren, J.L., Krajewski, S., Reed, J.C., Tilly, J.L., Isaacson, K.B., 1997. Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax. J. Clin. Endocrinol. Metab. 82, 2738–2746. Van Herreweghe, F., Festjens, N., Declercq, W., Vandenabeele, P., 2010. Tumor necrosis factor-mediated cell death: to break or to burst, that’s the question. Cell. Mol. Life Sci. 67, 1567–1579. Vaskivuo, T.E., Stenback, F., Karhumaa, P., Risteli, J., Dunkel, L., Tapanainen, J.S., 2000. Apoptosis and apoptosis-related proteins in human endometrium. Mol. Cell. Endocrinol. 165, 75–83. Watanabe, H., Kanzaki, H., Narukawa, S., Inoue, T., Katsuragawa, H., Kaneko, Y., Mori, T., 1997. Bcl-2 and Fas expression in eutopic and ectopic human endometrium during the menstrual cycle in relation to endometrial cell apoptosis. Am. J. Obstet. Gynecol. 176, 360–368. Wei, P., Jin, X., Tao, S.X., Han, C.S., Liu, Y.X., 2005. Fas, FasL, Bcl-2, and Bax in the endometrium of rhesus monkey during the menstrual cycle. Mol. Reprod. Dev. 70, 478–484. Wood, G.A., Fata, J.E., Watson, K.L., Khokha, R., 2007. Circulating hormones and estrous stage predict cellular and stromal remodeling in murine uterus. Reproduction 133, 1035–1044. Yamashita, H., Otsuki, Y., Matsumoto, K., Ueki, K., Ueki, M., 1999. Fas ligand, Fas antigen and Bcl-2 expression in human endometrium during the menstrual cycle. Mol. Hum. Reprod. 5, 358–364. Yonehara, S., Ishii, A., Yonehara, M., 1989. A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J. Exp. Med. 169, 1747–1756. Youle, R.J., Strasser, A., 2008. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47–59.