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Ovary; |
Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
ABSTRACT
Low oxygen caused by a decreasing blood supply is known to induce various responses of cells, including apoptosis. The present study was conducted to examine whether low-oxygen conditions (hypoxia) induce luteal cell apoptosis in cattle. Bovine midluteal cells incubated under hypoxia (3% O2) showed significantly more cell death than did those incubated under normoxia (20% O2) at 24 and 48 h of culture, and had significantly lower progesterone (P4) levels starting at 8 h. Characteristic features of apoptosis, such as shrunken nuclei and DNA fragmentation, were observed in cells cultured under hypoxia for 48 h. Hypoxia increased the mRNA expressions of BNIP3 and caspase 3 at 24 and 48 h of culture. Hypoxia had no significant effect on the expressions of BCL2 and BAX mRNA. Hypoxia also increased BNIP3 protein, and activated capsase-3. Treatment of P4 attenuated cell death, caspase-3 mRNA expression, and caspase-3 activity under hypoxia. Overall results of the present study indicate that hypoxia induces luteal cell apoptosis by enhancing the expression of proapoptotic protein, BNIP3, and by activating caspase-3, and that the induction of apoptosis by hypoxia is partially caused by a decrease in P4 production. Because hypoxia suppresses P4 synthesis in bovine luteal cells, we suggest that oxygen deficiency caused by a decreasing blood supply in bovine corpus luteum is one of the major factors contributing to both functional and structural luteolysis.
apoptosis, corpus luteum, progesterone, signal transduction, hypoxia
Luteal regression is characterized by a decrease in progesterone (P4) production (functional luteolysis), followed by a decrease in luteal size (structural luteolysis), during which cells of the corpus luteum (CL) undergo apoptosis [1–5]. In the cow, luteolysis is initiated by uterine prostaglandin (PG) F2
released at the late luteal stage [6]. It can also be induced by injection of PGF2 at the mid-luteal stage [7]. During both spontaneous and PGF2-induced luteolysis, a decrease in luteal blood flow occurs in parallel with systemic P4 concentrations [8–11]. Furthermore, the oxygen content in the ovarian venous blood begins to decrease at the late luteal stage [10]. The above findings indicate that the low-oxygen condition (hypoxia) caused by the decreased blood supply is a characteristic part of the luteal environment during luteolysis. Hypoxia has been suggested to induce apoptosis in many cell types, including ovarian cells [12–19]. However, the importance of hypoxia during luteolysis has not received much direct attention other than our recent report [20], which demonstrated that hypoxia decreases P4 synthesis in bovine CL. Moreover, although there is some indirect evidence that hypoxia contributes to luteolysis [8–11, 21, 22], less is known about whether hypoxia can induce apoptotic cell death in CL.
Apoptosis is modulated by many intracellular regulators, such as BCL2 family proteins [23–26] and caspases (CASPs) [25–27]. BCL2 family proteins modulate the release of apoptogenic factors, such as cytochrome c, apoptosis-inducing factor, and Smac/Diablo, from mitochondria into the cytosol. Release of such factors is believed to be one of the most important determinants of whether apoptosis will proceed or not [26]. Among the BCL2 family proteins, BCL2 is known to protect cells from apoptosis, while BAX accelerates cell death [23–26, 28]. Recently, a proapoptotic member of the BCL2 family, BNIP3, which promotes apoptosis through heterodimerization with the antiapoptotic proteins, including BCL2 and BCL2L1 [29], has been demonstrated to mediate hypoxia-induced apoptosis in Chinese hamster ovary [12]. However, it is not known whether BNIP3 is expressed or functions in bovine CL. CASPs, a family of aspartic acid-specific proteases, activate a proteolytic cascade and rapidly induce cell death [27]. In bovine CL, BAX [3, 30, 31] and CASPs 3, 8, and 9 [30] have been demonstrated to be involved in induction of structural luteolysis in vivo, as well as in vitro in bovine luteal cells [32, 33]. We previously found that hypoxia reduced P4 synthesis in bovine luteal cells [20], and P4 has been shown to suppress luteal cell apoptosis in bovine CL [34, 35]. Thus, we hypothesized that hypoxia induces luteal cell death by attenuating P4 production, and by regulating BCL2 family proteins and CASP3 expression and/or their activities.
The present study was conducted to examine whether hypoxia induces apoptotic cell death in bovine CL. In order to investigate possible mechanisms of hypoxia-induced cell death in bovine luteal cells, we also examined the effect of hypoxia on the expression or activity of BCL2, BAX, BNIP3, and CASP3.
Ovaries with a midstage CL from Holstein cows were collected at a local abattoir within 10–20 min after exsanguination. Midstage CLs were identified by macroscopic observation of the ovary and uterus as described previously [36]. For cell culture experiments, the ovaries with CL were submerged in ice-cold physiological saline and transported to the laboratory.
Luteal tissue was enzymatically dissociated and luteal cells were cultured as described previously [37]. The luteal cells were suspended in a culture medium, Dulbecco modified Eagle medium and Ham F-12 medium (1:1 [v/v]; D8900; Sigma-Aldrich, Inc., St. Louis, MO) containing 5% calf serum (16170–078; Life Technologies, Inc., Grand Island, NY) and 20 µg/ml gentamicin (15750–060; Life Technologies). Cell viability was greater than 85% as assessed by trypan blue exclusion. The cells in the cell suspension consisted of about 70% small luteal cells, 20% large luteal cells, 10% endothelial cells or fibrocytes, and no erythrocytes.
The dispersed luteal cells were seeded at 2.0 x 105 viable cells in 1 ml in 24-well cluster dishes (3524; Costar, Cambridge, MA) for detection of lactate dehydrogenase (LDH) release, P4 production, and mRNA expression, or in 6-well cluster dishes (657160; Greiner Bio-One, Frickenhausen, Germany) for TUNEL and propidium iodide (PI) staining (with glass slides) and detection of CASP3 activity, or in an 80-cm2 culture flask (658175; Greiner Bio-One) for detection of BNIP3 protein expression, and cultured in a humidified atmosphere of 5% CO2 in air at 38°C in an N2-O2-CO2-regulated incubator (BNP-110; ESPEC Corp., Osaka, Japan). After 12 h of culture, the medium was replaced with fresh medium containing 0.1% BSA, 5 ng/ml sodium selenite, and 5 µg/ml transferrin, and the following experiments were carried out. The cell culture under conditions with different levels of O2 (3% or 20%) was described previously [20]. By using this system, we previously confirmed the presence of hypoxic conditions by observing an increase in hypoxia-inducible factor 1 alpha (HIF1A) protein under 3% O2 [20].
Time-Dependent Effect of Hypoxia on Cell Death and P4 Production
Luteal cells were incubated under commonly used (normal) culture atmosphere (20% O2, 5% CO2, 75% N2) or low oxygen concentration (hypoxia) (3% O2, 5% CO2, 92% N2) for 4, 8, 12, 24, and 48 h. Conditioned media were collected and stored at –30°C until assayed for cytotoxicity and P4.
Effect of Hypoxia on DNA Fragmentation
Luteal cells were incubated under normal culture atmosphere (20% O2) or hypoxia (3% O2) for 48 h. After the culture, the cells were washed twice with 1 ml of PBS (05193; Seikagaku Corporation, Tokyo, Japan). The cells were fixed for 1 h at room temperature in PBS containing 4% paraformaldehyde, followed by two washes in PBS before permeabilization with 0.5% Triton X-100 (Bio-Rad) in PBS for 20 min. Cells were then briefly washed twice in PBS. The cells were incubated in 30 µl of fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase (TUNEL reagents; 8445; MBL, Nagoya, Japan) for 1 h at 37°C in a dark moist chamber. After the TUNEL reaction, the cells were washed twice in PBS and once in PBS containing 0.0002% PI (P4170; Sigma), then the cells were washed three times in PBS and stored in the dark at 4°C. The cells were observed under fluorescent illumination using a 470-nm excitation filter and a 515-nm absorption filter for fluorescein isothiocyanate and a 545-nm excitation filter and a 610-nm absorption filter for PI. A portion of cultured cells (200 cells) from each replication was used to calculate the percentage of TUNEL-positive cells. The percentage of TUNEL-positive cells was calculated by dividing the number of TUNEL-positive cells by the number of total cells counted under PI staining.
Effect of Hypoxia on BCL2, BAX, BNIP3, and CASP3 mRNA Expressions
Luteal cells were incubated under normal culture atmosphere (20% O2) or hypoxia (3% O2) for 24 and 48 h. After the culture, total RNA was extracted for determination of BCL2, BAX, BNIP3, and CASP3 mRNA expressions.
Effect of Hypoxia on BNIP3 Protein Expression
Luteal cells were incubated under normal culture atmosphere (20% O2) or hypoxia (3% O2) for 24 and 48 h. The cultured cells were scraped and placed in ice-cold homogenization buffer (25 mM Tris-HCl, 300 mM sucrose, 2 mM EDTA, Complete [protease inhibitor cocktail, 1697498; Roche Diagnostics GmbH, Mannheim, Germany], pH 7.4), and then frozen in liquid nitrogen and stored at –80°C until BNIP3 protein analysis by Western blotting.
Effect of Hypoxia on CASP3 Activity
Luteal cells were incubated under normal culture atmosphere (20% O2) or hypoxia (3% O2) for 24 and 48 h. After the culture, the cells were washed three times in PBS, and CASP3 activity was measured using a commercially available CASP3 colorimetric assay kit (CASP-3-C; Sigma) according to the instructions of the manufacturer (http://www.sigmaaldrich.com/sigma/bulletin/casp3cbul.pdf).
Effect of P4 on Hypoxia-Induced Apoptosis
Luteal cells were treated with P4 (0.5, 2.0, or 5.0 µg/ml) under normal culture atmosphere (20% O2) or hypoxia (3% O2) for 48 h. The concentration of P4 (0.5–2.0 µg/ml) was determined based on the P4 concentration in the ovarian venous blood in cattle [10]. The conditioned media, total RNA, and the cultured cells were collected and stored until analysis of cytotoxicity, BNIP3 and CASP3 mRNA expressions, BNIP3 protein expression, or CASP3 activity, as described for the above experiments.
Cytotoxicity was determined by measuring LDH in the conditioned media using a commercially available assay (1644793; Roche). The conditioned media (200 µl) were incubated with buffer containing NAD+, lactate, and tetrazolium for 30 min at room temperature. LDH converts lactate to pyruvate, generating NADH. The NADH then reduces tetrazolium (yellow) to formazan (red), which was detected by absorbance at 490 nm. The cells treated with 1% Triton X-100 were prepared for the positive control of LDH release from the cells according to the manufacturer's directions. Cytotoxicity was expressed as a percentage of the absorbance of the positive control in each culture period.
Concentrations of P4 were determined directly from the cell culture media with an enzyme immunoassay, as described previously [38]. The standard curve ranged from 0.391 to 100 ng/ml, and the median effective dose (ED50) of the assay was 4.5 ng/ml. The intra- and interassay coefficients of variation were 5.7% and 8.6%, respectively.
RNA Isolation and cDNA Synthesis
Total RNA was prepared from cultured luteal cells using TRIZOL Reagent (15596–026; Invitrogen, Carlsbad, CA) according to the manufacturer's directions. Total RNA (1 µg) was reverse transcribed using a ThermoScript RT-PCR system (11146–016; Invitrogen).
Real-Time Polymerase Chain Reaction (PCR)
Gene expression was measured by real-time PCR using a thermal cycler (Model MX3000; Stratagene, La Jolla, CA) and the QuantiTect SYBR Green PCR system (Qiagen GmbH, Hilden, Germany) starting with 1 ng of reverse-transcribed total RNA. Standard curves of sample cDNA were generated using serial dilutions (1:2 to 1:1000). The 18S ribosomal RNA expression was used as an internal control, and 20-bp primers with 50%–60% GC-contents were synthesized (Table 1). PCR conditions were: 95°C for 15 min, followed by 55 cycles of 94°C for 15 sec, 55°C for 30 sec, and 72°C for 30 sec. Use of the QuantiTect SYBR Green PCR system at elevated temperatures resulted in reliable and sensitive quantification of the PCR products with high linearity (Pearson correlation coefficient, r > 0.99).
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BNIP3 in the cultured bovine luteal cells was detected by Western blotting analysis. The cultured cells were lysed in 200 µl of lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 10% glycerol [G7757, Sigma], Complete, pH 7.4). Protein concentrations in the lysates were determined by the method of Osnes et al. [39], using BSA as a standard. The proteins were then solubilized in SDS gel loading buffer (50 mM Tris-HCl, 2% SDS [31607–94; Nacalai Tesque, Inc., Kyoto, Japan], 10% glycerol, 1% β-mercaptoethanol [137–06862; Wako Pure Chemical Industries, Ltd., Osaka, Japan], pH 6.8), and heated at 95°C for 10 min. Samples (50 µg protein) were subjected to electrophoresis on a 10% SDS-PAGE for 1 h at 200 V.
The separated proteins were electrophoretically transblotted to a 0.2 µm nitrocellulose membrane (LC2000; Invitrogen) at 250 mA for 3 h in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol, pH 8.3). The membrane was washed in TBS-T (0.1% Tween 20 in TBS [25 mM Tris-HCl, 137 mM NaCl, pH 7.5]), and incubated in blocking buffer (4% nonfat dry milk in TBS-T) overnight at 4°C. After blocking incubation, the membrane was cut into two pieces: one piece was used for detecting BNIP3 protein (21.5 kDa), and the other piece was used for β-actin (ACTB; internal standard; 42 kDa). The membranes were then incubated separately with a primary antibody specific to each protein: BNIP3 antibody (B7931; Sigma; 1:1000 in TBS-T) and ACTB antibody (A2228; Sigma; 1:4000 in TBS-T) for 1 h at room temperature, washed three times for 10 min in TBS-T at room temperature, incubated with secondary antibody (anti-mouse Ig, horseradish peroxidase-linked whole antibody produced in sheep [NA931; Amersham Biosciences Corp., Piscataway, NJ] 1:20 000 in TBS-T) for 1.5 h, and washed three times in TBS for 10 min at room temperature. The signal was detected by ECL Western Blotting Detection System (RPN2109; Amersham Biosciences Corp.).
The intensity of the immunological reaction in the cells was estimated by measuring the optical density in the defined area by computerized densitometry using NIH Image (National Institutes of Health).
All experimental data are shown as the mean ± SEM. The data of cytotoxicity and CASP3 activity are shown as a percentage of positive control and a percentage of control, respectively. The statistical significance of differences in cytotoxicity (see Figures 1 and 6), P4 production (see Figure 1), BNIP3 and CASP3 mRNA expressions (see Figures 7 and 8), BNIP3 protein expression (see Figure 7) and CASP3 activity (see Figure 8) was assessed by ANOVA followed by a Fisher protected least-squares difference procedure as a multiple comparison test. Statistical significance of differences in percentage of TUNEL-positive cells (see Figure 2B), BCL2, BAX, BNIP3, and CASP3 mRNA expressions (see Figure 3), BNIP3 protein expression (see Figure 4), and CASP3 activity (see Figure 5) was assessed by Student t-test.
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Time-Dependent Effect of Hypoxia on Cell Death and P4 Production
Hypoxia significantly induced luteal cell death at 24 and 48 h of culture (Fig. 1A; P < 0.05), whereas P4 production was significantly inhibited by hypoxia starting at 8 h of culture. Based on these results, further experiments were performed at 24 or 48 h of culture.
Effect of Hypoxia on DNA Fragmentation
Staining with PI showed that the nuclei of the cells cultured under hypoxic condition for 48 h were condensed and fragmented (Fig. 2A). Moreover, TUNEL staining indicated that DNA fragmentation occurred in most cells cultured under hypoxic condition for 48 h (Fig. 2, A and B; P < 0.01).
Effect of Hypoxia on BCL2, BAX, BNIP3, and CASP3 mRNA Expressions
The results of real-time PCR analysis showed that O2 concentration was not significantly related to the expressions of BCL2 and BAX mRNA (Fig. 3, A and B). On the other hand, BNIP3 and CASP3 mRNA expressions were markedly increased by hypoxia at 24 and 48 h of culture (Fig. 3, C and D, respectively; P < 0.05).
Effect of Hypoxia on BNIP3 Protein Expression
BNIP3-specific bands were expressed after 24- and 48-h incubation, regardless of O2 concentration (Fig. 4A). The intensities of the BNIP3-specific bands, after normalization to ACTB-specific bands, significantly increased after exposure of bovine luteal cells to hypoxic conditions for 24 and 48 h (Fig. 4B; P < 0.05).
Effect of Hypoxia on CASP3 Activity
The enzymatic activity of CASP3 in bovine luteal cells was significantly stimulated by culture under hypoxic conditions for 24 and 48 h (Fig. 5; P < 0.05).
Effect of P4 on Hypoxia-Induced Apoptosis
P4 attenuated cell death under hypoxia as well as under normoxia (Fig. 6; P < 0.05). P4 did not affect BNIP3 mRNA expression (Fig. 7A), whereas CASP3 mRNA expression was decreased by P4 (5.0 µg/ml) (Fig. 8A; P < 0.05). CASP3 activity was also inhibited by P4 at all concentrations tested (0.5–5.0 µg/ml; Fig. 8B; P < 0.05). On the other hand, BNIP3 protein expression was increased by P4 (2 µg/ml) under hypoxia (Fig. 7B; P < 0.05).
The present study demonstrated for the first time that reduced oxygen tension (hypoxia) induced luteal cell death in cattle. Moreover, the finding of shrunken nuclei and apoptotic bodies in cells cultured under hypoxia, and the finding that CASP3 was activated by hypoxia, indicate that hypoxia-induced bovine luteal cell death occurs by apoptosis. Both ovarian blood flow [10, 21] and intraluteal blood flow [11] have been demonstrated to decrease during luteolysis in cows. Thus, the present results provide strong evidence that hypoxia, caused by decreasing blood supply, induces apoptosis of the cells in the CL, resulting in structural luteolysis. Furthermore, since we have recently demonstrated that hypoxia markedly inhibited P4 production in bovine luteal cells [20], we propose that hypoxic conditions, induced by a decreased blood supply, is essential for the progression of the luteolytic cascade in cattle.
Hypoxia has been suggested to play some roles in luteolysis in ruminants, because ovarian blood flow decreases during luteolysis in ewes [8, 9] and cows [10, 11, 21]. Until recently, however, it has not been clarified whether hypoxia influences P4 synthesis or apoptosis of luteal cells, without a recent report, in primate luteal cells [40]. In the ewe, vascular occlusion occurred following the sloughing of endothelial cells into the lumina of small blood vessels during luteolysis, suggesting that hypoxia caused by the vascular occlusion induces apoptosis of cells in CL [22]. In the cow, we have recently demonstrated that hypoxia inhibits P4 synthesis in bovine luteal cells by attenuating P450scc activity, suggesting that hypoxia accelerates functional luteolysis [20]. In the present study, hypoxia increased apoptotic cell death of bovine luteal cells, suggesting that hypoxia facilitates structural luteolysis. Furthermore, the present time-course studies revealed that hypoxia induced a significant decrease of P4 production before it induced a significant increase in luteal cell death (i.e., P4 production was decreased by hypoxia starting at 8 h of culture, while luteal cell death was not induced by hypoxia until 24 h of culture) (Fig. 1). An in vivo study [2] also showed that apoptosis first appeared after the serum P4 concentration decreased. Together, these findings suggest that, during luteolysis in cattle, hypoxia caused by a decreasing blood supply causes a decrease in P4 synthesis (functional luteolysis), and then induces apoptosis of luteal cells (structural luteolysis).
We recently showed that P4 production in bovine luteal cells decreased when the cells were cultured under hypoxic conditions, suggesting that hypoxia is one of the causes of functional luteolysis [20]. P4 has been suggested to act as a suppressor of cell death in bovine luteal cells [34, 35]. Moreover, since treatment with P4 (albeit at high concentrations: 2.0–5.0 µg/ml) attenuated hypoxia-induced luteal cell death in the present study, the hypoxia-induced cell death is partly due to decreased P4 production by hypoxia. We recently showed that a specific P4 receptor antagonist increased the mRNA expression and activity of CASP3 in bovine luteal cells, and suggested that P4 suppresses apoptosis via inhibiting CASP3 action [35]. Furthermore, we show in the present study that P4 production in the cells was significantly reduced by hypoxia, and that addition of P4 (0.5–5.0 µg/ml) under hypoxia down-regulated mRNA expression and activity of CASP3. Thus, the above findings suggest that hypoxia increases CASP3 mRNA expression and activity by inhibiting P4 production, resulting in luteal cell death by apoptosis. In addition to this mechanism, the other mechanisms or factors are thought to contribute to hypoxia-induced luteal cell apoptosis, because the P4 concentrations (0.5–5.0 µg/ml) used in the present study were relatively high (similar to the P4 concentrations in ovarian venous blood: 0.1–2.0 µg/ml [10]), and because the effects of P4 were significant, but not strong enough to suppress the cytotoxic effect of hypoxia to the control level. We are presently searching for other luteal cell-derived survival factors that are suppressed by hypoxia.
Under hypoxic conditions, HIF1A has been suggested to induce apoptosis via two mechanisms [16, 41, 42]. In one mechanism, HIF1A associates with p53, stabilizes p53, and induces apoptosis via BAX overexpression, while, in the other mechanism, HIF1A binds to aryl hydrocarbon receptor nuclear translocator (ARNT, also called HIF1B) and induces apoptosis via overexpression of BNIP3 and BNIP3L, a BNIP3 homologue. In the present study, hypoxia significantly increased the mRNA and protein levels of BNIP3, but did not affect the mRNA levels of BCL2 and BAX, suggesting that BNIP3 is more sensitive to hypoxia than BAX and BCL2 in luteal cells. Furthermore, because we have recently found that 3% O2 induces HIF1A protein accumulation in cultured bovine luteal cells [20], we hypothesized that hypoxia induces apoptosis at least in part via the HIF1A-BNIP3 mechanism in bovine luteal cells. On the other hand, severe hypoxia has been demonstrated to induce overexpression of BAX in human umbilical vein endothelial cells and mouse embryonic stem cells [16], and to suppress BCL2 expression in cultured human aortic endothelial cells [14]. Thus, it is possible that the low oxygen condition used in the present study (3% O2 for 48 h) was not severe enough to induce BAX overexpression or suppress BCL2 expression in bovine luteal cells.
Interestingly, BNIP3 protein expression was unexpectedly up-regulated by P4 treatment under hypoxia. The physiological significance of this phenomenon is unclear. Cell death and CASP3 activity, as well as CASP3 mRNA expression, were slightly but significantly attenuated by P4 under hypoxia. These findings suggest that inhibition of P4 production is not needed for inducing apoptosis via BNIP3 under hypoxia. Based on the above findings, we suppose that hypoxia induces apoptosis via two mechanisms. One is illustrated in the earlier paragraph stating that hypoxia induces apoptosis via the HIF1A-BNIP3 mechanism. The other is that hypoxia activates CASP3 by inhibiting P4 synthesis, resulting in induction of apoptosis (see Fig. 9).
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The overall results indicate that hypoxia induces luteal cell death by increasing BNIP3 protein expression and CASP3 activity, and suggest that these effects of hypoxia are partly induced by inhibiting P4 production in bovine luteal cells. Furthermore, since hypoxia inhibits P4 production by cultured bovine luteal cells [20], we suggest that an oxygen deficiency caused by a decreasing blood supply is necessary for the progression of both functional and structural luteolysis in cattle.
FOOTNOTES
1Supported by Grants-in-Aid for Scientific Research 18380166 and 18658114 of the Japan Society for the Promotion of Science (JSPS). R.N. is a JSPS Research Fellow funded by grant 03589. 
Correspondence: 2Kiyoshi Okuda, Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka 1-1-1, Okayama 700-8530, Japan. FAX: 81 86 251 8333; e-mail: kokuda{at}cc.okayama-u.ac.jp
Received: 9 June 2007.
First decision: 14 July 2007.
Accepted: 16 November 2007.
REFERENCES
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  A. Bowolaksono, R. Nishimura, T. Hojo, R. Sakumoto, T. J. Acosta, and K. Okuda Anti-Apoptotic Roles of Prostaglandin E2 and F2alpha in Bovine Luteal Steroidogenic Cells Biol Reprod, August 1, 2008; 79(2): 310 - 317. [Abstract] [Full Text] [PDF]
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