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Regular Article |
a Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia 30310
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, clinostat, corpus luteum function, mitochondria, ovary, pregnancy, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Lewis et al. [6] have reported that the human lymphoblastoid cell line placed on the space shuttle shows cytoskeletal alteration, growth retardation, and metabolic changes concomitant with increased apoptosis. Recently, Sarkar et al. [7] have also shown that culture in the vector-averaged gravity (simulated microgravity) under clinostat rotation results in apoptosis of osteoblastic cells.
Apoptosis results from the action of a genetically encoded suicide program that leads to a series of characteristic morphological and biochemical changes [8]. These changes include cell shrinkage, chromatin condensation, DNA fragmentation, mitochondrial depolarization, and activation of apoptosis-related enzymes [9–11]. Apoptosis can be triggered by a variety of external stimuli such as Fas ligand, free radicals, chemotherapeutic drugs, and UV radiation [12]. Generally, external signals passing through the cell membrane induce the production and activation of apoptosis-related proteins by various series of signal transduction cascade. It is known that many of these proteins, such as bcl-2 family proteins and cytochrome c, are located in the mitochondria [11, 13]. When apoptotic signals stimulate cells, the membrane potential of mitochondria changes, and cytochrome c is released from mitochondria to cytoplasm, resulting in the activation of caspases [14]. However, little is known about the mechanism by which changes in physical or mechanical forces, including gravity, induce apoptosis.
In our studies, the microgravity environment without the free fall of the cells was attained using the vector-free, single-axis horizontal clinostat. The clinostat model system (clinostat rotation), a ground-based method for providing a vector-averaged reduction in the apparent gravity on the cell culture, has been used to produce the simulated microgravity environment for more than 10 yr [15, 16]. Clinostat randomizes the orientation of the gravitational vector and is an established model for studying various mammalian cells under microgravity.
In the present study, we hypothesized that microgravity conditions generated by clinostat rotation induce apoptosis of luteal cells by altering the mitochondrial membrane potential, leading to a fall in the synthesis of progesterone. To test the hypothesis, we evaluated apoptosis in the luteal cells cultured in microgravity conditions generated by clinostat rotation. Further, we have taken the approach of detecting the mitochondrial membrane potential in the luteal cells with JC-1 dye to assess whether apoptosis brought about by clinostat is due to mitochondrial damage.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Medium 199 was obtained from Gibco BRL (Grand Island, NY). Collagenase (type 4) was purchased from Worthington Biochemical Corporation (Freehold, NJ). DNase 1 and dispase 2 were obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Fetal bovine serum was purchased from Hyclone Laboratories (Logan, UT). Rat tail collagen (type 1) was obtained from Collaborative Biomedical Products (Bedford, MA). Methoxyflurane (Metofane; Mallinckrodt Veterinary, Inc., Mundelein, IL) anesthesia was obtained through our institutional animal care facility. The progesterone radioimmunoassay kit was purchased from Diagnostics Systems Laboratories, Inc. (Webster, TX), the protein assay kit from Bio-Rad Laboratories (Hercules, CA), the Comet assay kit from Trevigen, Inc. (Gaithersburg, MD), and the in situ apoptosis detection kit (Apoptag) from Intergen Company (Purchase, NY). The remaining chemicals were purchased from Sigma Chemical Company (St. Louis, MO), unless otherwise stated.
Animals
Timed-pregnant Sprague-Dawley rats were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). They were housed in our institutional animal facility at a temperature of 23–25°C, and exposed to a photoperiod of 14L:10D (lights-on at 0500 h; lights-off at 1900 h). Purina rat chow (Richmond, IN) and tap water were accessible ad libitum. The day of insemination, identified by a sperm plug, was designated as Day 1 of pregnancy. All procedures involving animals were conducted only after approval by our Institutional Animal Care and Use Committee in accordance with the principles and procedures of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Collection of Corpora Lutea and Separation of Luteal Cells
On Day 8 of pregnancy, the ovaries were removed while the rats were under Metofane anesthesia. The corpora lutea were separated, cleaned of any adhering follicles, and placed in Medium 199 containing 2.2 g/L of sodium bicarbonate previously gassed with 95% O2 and 5% CO2. After all the corpora lutea were dissected, the incubation medium was changed to Hanks balanced salt solution (HBSS) with 0.1% BSA, but without calcium and magnesium, to which collagenase (50 U/ml), dispase (2.4 U/ml), and DNase (200 U/ml) were added. Four incubations lasting 30 min each were carried out in a shaking water bath at 37°C. The corpora lutea were then placed in 0.2% EDTA solution containing 0.1% BSA and gently stirred for 15 min. The tissue suspension was then centrifuged at 200 x g for 5 min, and then the EDTA solution was replaced with HBSS containing 0.1% BSA but without calcium and magnesium. The corpora lutea were dissociated by repeatedly resuspending the tissue/cells; the resulting mixed population of luteal cells were then counted in a hemocytometer using the standard Trypan blue dye-exclusion method given with the manufacturer's protocol. The cell suspension was centrifuged at 200 x g for 5 min, and then resuspended in Medium 199 containing 25 mM Hepes buffer, 100 IU of penicillin, and 100 µg of streptomycin/ml, 50 µg of gentamycin/ml, and 10% fetal bovine serum, and used further for primary culture of luteal cells.
Clinostat Culture of Luteal Cells
Luteal cells (0.5 x 106 per slide) were plated to collagen-coated chamber slides (Nalgene Nunc International, Roskilde, Denmark) 24 h before subjecting them to microgravity conditions. At 24 h after preculture for the attachment of cells to the surface of the chamber slides, the chambers were filled with the medium (previously gassed with 95% O2 and 5% CO2), capped tightly, and then placed in the clinostat (catalog number 099ATC108; Glas-Col, Terre-Haute, IN) at a speed of 30 rpm (Day 0). For stationary control, chamber slides were placed in the same incubator alongside the clinostat. Medium was changed daily at the same time for 5 days, and daily progesterone level was assayed.
Progesterone Assay
Progesterone levels in the cell culture media were determined using a radioimmunoassay kit. The sensitivity of the assay was 0.12 ng/ml. The coefficient of interassay and intraassay variations were 3.9% and 4.6%, respectively.
Protein Assay
The protein concentration of the attached cell extracts was estimated by a modified Lowry method (DC protein assay kit; Bio-Rad Laboratories, Inc., Hercules, CA).
Propidium Iodide Staining
To assess whether death of cells cultured in the clinostat occurred by necrosis, we performed staining with propidium iodide without fixation. After culturing for 2 days, 10 µl of propidium iodide solution (0.05% w/v propidium iodide in PBS) was added to the media, and the stained cells were observed under a fluorescent microscope. Cells with red nuclei were considered necrotic. For quantifying necrotic cells, 300–500 cells in 5 random fields were counted for each slide, and the numbers of necrotic cells were expressed as percentages of the total cell population. Data from 3 experiments were combined and expressed as means ± SEM.
Single-Cell Gel Electrophoresis
After harvesting cells cultured on slides, cells were combined with low-melting agarose at 42°C and placed on the CometSlide. The slide was placed at 4°C in the dark for 10 min and left in prechilled lysis solution at 4°C for 30 min. After tapping off excess buffer, the slides were placed in alkali buffer (1.2% NaOH, 1 mM EDTA) for 60 min at room temperature in the dark. Then, the slides were washed twice in TBE buffer (0.1 M Trizma-base, 0.1 M boric acid, 3 mM EDTA) for 5 min, and transferred onto a gel tray submerged in TBE buffer. Electrophoresis was performed at 20 volts for 20 min. After electrophoresis, excess TBE was tapped off the slide, and then 50 µl of SYBR Green was added onto agarose on the slide. The Comet tails, which are characteristic of apoptosis, were observed under a fluorescent microscope.
TUNEL Assay
The cells cultured on the slide were fixed with 4% neutral buffered formalin (4% v/v formalin solution in Tris buffer) and postfixed in ethanol:acetic acid (2:1) for 5 min at -20°C. Fixed cells were incubated with an equilibrium buffer for 5 min using the ApopTag kit and then treated in reaction buffer with 10 units of terminal deoxynucleotidyl transferase and 1 nmol deoxyuridine triphosphate-digoxigenin at 37°C for 2 h. The reaction was terminated by adding stop/wash buffer and then washed twice with Tris buffer. Antidigoxigenin-horseradish peroxidase was added and reacted at 37°C for 30 min. After washing with distilled water, nuclei were counterstained with hematoxylin, and apoptosis in the cells was observed under a light microscope. Cells with deep brown nuclei were considered apoptotic. For quantifying apoptotic cells, apoptotic and total cells were counted in 5 random fields scoring between 300 and 500 cells, and the numbers of apoptotic cells were expressed as percentages of the total cell population. Data from 3 experiments were combined and expressed as means ± SEM.
Staining of Mitochondria with Active Membrane Potential
To investigate how the microgravity conditions induce apoptosis, cultured cells were stained with JC-l dye, which can specifically detect mitochondria with active membrane potential. Because JC-1 dye is sensitive to mitochondrial membrane potentials, it stains the mitochondria with a membrane potential less than 140 mV in green-yellow. At membrane potentials greater than 140 mV, JC-1 forms J-aggregate, which emits a red signal under a fluorescent microscope. After culturing for 48 h, luteal cells in the clinostat and the control were stained with 50 µl of JC-1 (10 µg/ml) in the medium for 10 min in an incubator. Cells were rinsed in dye-free culture medium and observed under a fluorescence microscope.
Statistical Analysis
ANOVA followed by Scheffé correction was used to evaluate the overall differences in means among the groups. A paired Student t-test with correlated samples was used to compare differences between the control and the clinostat. A P value < 0.05 was considered statistically significant. All results are presented as means ± SEM of combined data from the replicate experiments (n = 3 to 6).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Progesterone production by the cultured luteal cells was not different between the control and the clinostat 1 day after culture. However, progesterone levels in the control increased on Day 2 and continued to increase on Day 3. From Day 3 to Day 5 the progesterone levels remained at a high level, whereas its levels in the clinostat did not increase on Day 2 and remained low through Day 5 (Fig. 1).
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Protein Concentration
The protein concentration of attached cells on Day 1 was not different between the control and the clinostat. However, the protein concentration in the clinostat decreased on Day 2 and remained low through Day 5, showing a difference compared with that in the control (Fig. 2). Therefore, we decided to use Day 2, which showed significant differences in both progesterone level and protein concentration, as the time point to make comparisons of cell death between the clinostat and the control.
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Evaluation of Cell Death
We observed that the cultured luteal cells experienced shrinking and fragmentation on Day 2. There were more shrunken cells in the clinostat than in the control (Fig. 3). To assess whether these shrunken cells died by necrosis, cells cultured for 2 days were stained with propidium iodide without fixation, and were observed under a fluorescent microscope (Fig. 4A). The percentage of cells with red nuclei stained with propidium iodide was less than 10% in both the control and the clinostat on Days 2 and 5 (Fig. 4B). In addition, we found that the fragmented and shrunken cells did not stain with propidium iodide. These data suggested that the shrunken cells were not the products of necrosis. Therefore, we performed the Comet and TUNEL assays to determine whether apoptosis occurred in the cultured cells.
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Qualitative Apoptosis Assessment by Comet Assay
Figure 5 shows comets of the cultured luteal cells in the control and in the clinostat after electrophoresis. Most of the nuclei in the control appeared nonapoptotic, displaying negligible tails. In the clinostat, however, we observed a number of comets that were characteristic of apoptosis displaying long, puffy tails.
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Quantitative Apoptosis Assessment by TUNEL Assay
Shrunken and blebbed-shape cells, which characterize apoptosis, were stained deep brown (arrowheads) using TUNEL (Fig. 6A). The luteal cells cultured for 2 days appeared to have more apoptotic cells in the clinostat compared with the control, showing a significant difference (P < 0.05; Fig. 6B). Individual data points for each set of experiments showed clear-cut differences in terms of more apoptotic cells in the clinostat compared with the control (Fig. 6B, inset).
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Mitochondrial Membrane Potential in Cells
When cultured cells were stained with JC-1 dye and examined under a fluorescent microscope, most of cells in the control on Days 2 and 5 showed a great number of active mitochondria, which were evenly distributed throughout the cell (Fig. 7). On the other hand, many cells in the clinostat displayed fewer active mitochondria, which were distributed to the outer edge of cell or concentrated around the nucleus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, it has been reported that the cells flown in space or subjected to clinostat rotation show increased apoptosis by altered organization of their cytoskeleton [6, 7]. Based on this, we hypothesized that a decrease in progesterone levels produced by the luteal cells in the clinostat may be due to apoptosis induced by microgravity conditions. To investigate whether apoptosis occurred in the luteal cells cultured in the clinostat, we observed morphology of the luteal cells under light microscope and detected apoptosis in these cells by using various methods. Under light microscope, we observed that there were more shrunken and dark cells in the clinostat than in the control on Day 2. To confirm whether these shrunken cells were a result of apoptosis or necrosis, cells were stained with propidium iodide without fixation. Generally, apoptotic cells do not stain with propidium iodide without fixation, because the plasma membrane remains intact during apoptosis. However, necrotic cells lose the integrity of their plasma membranes in the process of cell death and are readily stained with propidium iodide [21]. In the present study, we found that most dark and shrunken cells did not stain with propidium iodide, suggesting that the shrunken cells did not undergo necrosis. To confirm this observation that apoptosis occurred in the shrunken cells, we performed the Comet assay to detect apoptosis at the single cell level and the TUNEL assay to quantify these data.
The qualitative evaluation of apoptosis in the clinostat by the Comet assay was substantiated by the quantitative analysis of apoptosis by TUNEL assay. Comet assay, a single-cell gel electrophoresis assay, is a simple and effective method for evaluating DNA damage to cells [22]. After electrophoresis of cultured cells embedded in low-melting agarose and staining with fluorescence dye, live cells show intact nuclei, whereas apoptotic cells show a long, puffy tail from the nucleus [23]. Our results demonstrate that most of the cells in the control possessed intact nuclei with negligible tails, whereas in the clinostat, many of the cells displayed comets with long, puffy tails. Our results from the TUNEL assay demonstrate that the luteal cells cultured for 2 days showed more apoptotic cells in the clinostat compared with those in the control. However, the percentage of necrotic cells stained with propidium iodide was less than 10% in both the control and the clinostat. From the TUNEL assay and propidium iodide staining results, we concluded that the luteal cells cultured in the clinostat undergo a significant amount of apoptosis, but negligible amount of necrosis.
Our results further suggest that microgravity conditions could result in dysfunction of mitochondria, thereby inhibiting cholesterol transport and function of steroidogenic enzymes, leading to suppressed production of progesterone. We performed a total protein assay to evaluate whether a decrease in the number of cells attached on the slides due to cell death is correlated to a drop in progesterone levels. Similar to the progesterone levels, the protein level on Day 1 was no different between the control and the clinostat, but its level in the clinostat decreased on Day 2, showing a significant difference compared with those in the control. However, the reduction rate of progesterone levels in the clinostat was more than twice that of total protein levels after Day 3. This difference in reduction rates suggests that a fall in progesterone levels may be due to not only cell death but also due to other reasons such as the dysfunction of enzymes involved in steroidogenesis. Mitochondria have key enzymes associated with steroidogenesis such as P450 side chain cleavage (P450scc), which converts cholesterol to pregnenolone [24]. Furthermore, mitochondria consist of transmembrane proteins related to the permeability of the mitochondrial membrane and cholesterol transport, such as peripheral benzodiazepine receptor (PBR) and steroidogenic acute regulatory protein (StAR) [25, 26]. We have recently demonstrated an increase in the amount of lipid droplets in luteal cells exposed to microgravity conditions in a clinostat, supporting the notion that microgravity conditions inhibit steroidogenesis [5]. We have also reported that the increased quantity of lipid droplets in luteal cells is due to the disruption of progesterone production just after the point of cholesterol synthesis or uptake in the biosynthetic pathway (i.e., cholesterol is abundant in an esterified form) [27].
To assess how the microgravity conditions induce dysfunction of mitochondria, luteal cells were stained with JC-1 dye, which can specifically indicate the loss of the mitochondrial potential [28]. The dye readily enters live cells and fluoresces bright red in its multimeric form within active mitochondria. In the apoptotic cells, the mitochondrial membrane potential collapses, and the JC-1 dye fails to express its multimeric form within the mitochondria. In these cells, JC-1 dye remains in the cytoplasm as green J-monomeric form. Thus, apoptotic cells showing primarily green fluorescence are easily differentiated from live cells. In the present study, most of the cells cultured in the control showed a great number of red mitochondria, which were evenly distributed throughout the cell. To the contrary, cells cultured in the clinostat displayed fewer red mitochondria, which were distributed to the outer edge of the cell or around the nucleus. Although we could not quantify the number of cells because of technical difficulties, the loss of mitochondrial membrane potential was obvious under microgravtiy conditions. Several recent papers have reported that the loss of the mitochondrial membrane potential induces apoptosis [29, 30]. The change in mitochondrial permeability is an important event in the apoptotic process wherein the electrochemical gradient across the mitochondrial membrane collapses. The collapse is believed to occur by the formation of pores in the mitochondrial membrane thereby releasing cytochrome c from the mitochondria to cytoplasm, which results in the induction of apoptosis [11, 14]. In addition, if the mitochondria are not functioning properly, mitochondrial proteins such as bcl-2, bax, PBR, StAR, and P450scc also lose their function, inducing apoptosis, and thus inhibiting steroidogenesis [25, 31, 32]. Further, it is possible that mitochondrial dysfunction of luteal cells cultured under microgravity conditions may result from a change in the structural integrity of cytoskeleton, because movement and distribution of mitochondria in the cells are associated with microtubules [33]. Disruption of cytoskeletal integrity has been suggested as a gravity-sensing mechanism in single cells [34, 35]. Collectively, these findings support the hypothesis that inactivation of mitochondria by conditions causing cytoskeletal collapse leads to an increase in apoptosis of the luteal cells under microgravity. This is being currently investigated in our laboratory using the primary luteal cell culture model described in this study.
In summary, the results of the present study demonstrate that microgravity conditions generated in the clinostat induce apoptosis and suppress the production of progesterone by the luteal cells of pregnant rats. The induction of apoptosis and inhibition of progesterone production by the luteal cells may be due to the dysfunction of mitochondria induced by microgravity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was presented at the 15th annual meeting of the American Society for Gravitational and Space Biology in Seattle, Washington, November 10–13, 1999, abstract 107. This study was supported by grants NAG9-963 and NCC953 from the National Aeronautics and Space Administration, and by grant SO6-GMO8248 from the National Institutes of Health. 
2 Correspondence: Rajagopala Sridaran, Department of Physiology, Morehouse School of Medicine, 720 Westview Drive, Atlanta, GA 30310-1495. FAX: 404 752 1045; sridaran{at}msm.edu
Accepted: October 25, 2001.
Received: June 19, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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