- Email: [email protected]
Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by l -proline
Cryobiology xxx (2015) 1e6
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline Xiaoyun Wang, Youzhi Wang* China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 September 2015 Received in revised form 20 November 2015 Accepted 30 November 2015 Available online xxx
Freeze-drying is one of the most effective methods to preserve fungi for an extended period. However, it is associated with a loss of viability and shortened storage time in some fungi. This study evaluated the stresses that led to the death of freeze-dried Mucor rouxii by using cell apoptotic methods. The results showed there were apoptosis-inducing stresses, such as the generation of obvious intracellular reactive oxygen species (ROS) and metacaspase activation. Moreover, nuclear condensation and a delayed cell death peak were determined after rehydration and 24 h incubation in freeze-dried M. rouxii via a propidium iodide (PI) assay, which is similar to the phenomenon of cryopreservation-induced delayed-onset cell death (CIDOCD). Then, several protective agents were tested to decrease the apoptosis-inducing stresses and to improve the viability. Finally, it was found that 1.6 mM L-proline can effectively decrease the nuclear condensation rate and increase the survival rate in freeze-dried M. rouxii. In conclusion: (1) apoptosis-inducing factors occur in freeze-dried M. rouxii. (2) ROS and activated metacaspases lead to death in freeze-dried M. rouxii. (3) L-proline increases the survival rate of freeze-dried M. rouxii. © 2015 Elsevier Inc. All rights reserved.
Keywords: Apoptosis Cryopreservation Freeze-drying L-proline Viability Mucor rouxii
1. Introduction Freeze drying is a convenient technique favored by many large culture collections. Freeze-drying of an isolate can be performed by freezing and drying a suspension of the fungus in its nutritive medium using a freeze dryer in conjunction with a vacuum apparatus. Ampoules with freeze-dried fungi are sealed to prevent damage of lyophilized material as a result of contact with air. With this technique, it is common for the fungal conidia to survive [5,25]. Moreover, storage by freeze-drying is one of the most suitable methods for maintaining viable and stable fungal cultures for 20e40 years. It is a convenient and successful method for preserving sporulating fungi and is ideal for the distribution of the organisms. However, there are some restrictions, such as loss of viability and a shortened storage time in some fungi. Optimization of the freeze-drying protocol included selection of the candidate fungus, optimizing physiological growth conditions and age, standardizing the protectant type and concentration, optimizing the pre-freezing
* Corresponding author. E-mail address: [email protected] (Y. Wang).
method, freeze-drying run and extent of drying, and choosing the rehydrant and the extent of rehydration [26]. As far as we know, this is the first study on apoptosis in the field of freeze-driedpreserved fungi. The field of cryopreservation has a long and successful history of in depth study and progress. In 1998, several independent groups reported the direct involvement of apoptotic and necrotic cell death following cryopreservation [1]. Many studies have identified diverse molecular-based cellular responses to cryopreservation and have further demonstrated significant improvements in cell survival through the inhibition of apoptosis. A substantial volume of studies have described the modulation of cell death through the use of various protease inhibitors, free radical scavengers, media formulations, and other novel compounds [1,3]. However, understanding of the pathway activation, progression, control and the downstream effect on cell apoptosis during cryopreservation remains in its infancy. Modulation studies, such as targeted apoptotic control (TAC), have shown promise in furthering our understanding of the activation pathways and are proving to be a critical next step in the evolution of cryopreservation sciences [3]). Therefore, the aims of this study were to answer the following questions about freeze-dried Mucor rouxii: 1) Are apoptosis-
http://dx.doi.org/10.1016/j.cryobiol.2015.11.006 0011-2240/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
2
X. Wang, Y. Wang / Cryobiology xxx (2015) 1e6
inducing factors involved in the death of freeze-dried M. rouxii? 2) Do apoptosis inhibitors such as caspase inhibitor ZVAD-fmk and ROS-scavengers have a protective effect on freeze-dried M. rouxii? These questions have general importance to the understanding of fungal death during freeze-drying and special importance for the use of freeze-dried M. rouxii in many applications. 2. Materials and methods 2.1. Strain and culture conditions M. rouxii strain CGMCC 3.2545, from the China General Microbiological Culture Collection Center, was used in this study. The fungus was cultivated on PDA (20% potato extract, 2% dextrose, 2% agar) medium or Czapek-Dox medium (0.2% sodium nitrate, 0.1% potassium phosphate dibasic, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferric sulfate, 3% sucrose) for 7e8 d at 28 C. 2.2. Freeze-drying of M. rouxii Fungal cultures at 7 days old were selected for freeze drying according to the standard procedure in CGMCC. Approximately 4 ml of sterile skimmed milk was added to a slanted culture and the surface of the agar was gently scraped with a sterile blade to obtain a sporangiospore suspension, which was filtered through four layers of sterile lens wiping paper to remove hyphal fragments. The sporangiospore suspension was added to each ampoule which was then loosely plugged with a cotton swab and put in a 20 C refrigerator for pre-freezing. Sporangiospores were freeze-dried and then sealed under vacuum using a gas-oxygen torch and stored at 4 C. At least three ampoules were used in each of the following experiments. Freeze-dried M. rouxii sporangiospores (106 sporangiospores/mL) were rehydrated with sterile water and used for the following tests; the sporangiospores before pre-freezing were used as a control. 2.3. Viability assay Dead or dying cells with disrupted membranes were detected microscopically using propidium iodide (PI), a nucleic-acid-binding fluorescent probe that is excluded by intact membranes of viable cells, as well as the procedure of Pinto et al. [21] with some modifications. After rehydration and incubation for 0 h, 12 h and 24 h at 25 C, cells were washed (2000 g for 3 min) and stained with 1 mg/ mL PI (Sigma, St. Louis, Missouri) for 5 min. After centrifugation, a sample of the cell precipitate for each treatment was viewed on a glass slide with a ZEISS Axioplan 2 fluorescent microscope using filter set 4 for PI (excitation wavelength 488 nm and emission wavelength 615 nm). The percentage of viable cells was determined as: [viable cells/(dead cells þ viable cells)] 100%. The dilution plate method was also used to test the survival rate of freeze-dried M. rouxii sporangiospores. Before pre-freezing, 0.2 ml M. rouxii spore suspension was diluted 104 times, and 0.1 ml was painted on PDA plates. After culturing in a 25 C incubator for approximately 17e19 h, colony-forming units (CFUs) were counted as NA. After freeze drying, M. rouxii sporangiospore powder was rehydrated, diluted, and painted on PDA plates as described. The CFUs were counted as NB. The survival rate was calculated by NB/NA 100%. 2.4. Fluorescence and light microscopy For each replicate of each treatment, at least 200 cells were examined with a ZEISS Axioplan 2 microscope (Germany) for
fluorescence applications and filter set 5 for light microscopy with differential interference contrast. All images were captured with an AxioCamMR5 camera and processed by Adobe PhotoShop 7.0. 2.5. Statistical analyses All tests were performed at least in triplicate. The results are expressed as the mean ± SD (standard deviation). The comparison between the controls and the freeze-dried M. rouxii that was treated with the caspase inhibitor and ROS scavengers was analyzed with a paired t-test using SPSS17.0. P < 0.05 was considered to indicate a statistically significant difference. 2.6. Apoptosis analysis Activated metacaspases were detected microscopically with CaspACE FITC-VAD-fmk In Situ Marker (Promega, Madison, USA) as described by Wu et al. [29]. After rehydration and incubation for 0 h, 1 h and 4 h at 25 C, the cells were washed and stained with FITC-VAD-FMK according to the manufacturer's instructions. Cells were washed and precipitated as above for fluorescent microscopy using filter set number 3 (excitation wavelength 488 nm and emission wavelength 530 nm). The intracellular production of reactive oxygen species (ROS) was detected with the oxidant-sensitive probe dichlorodihydrofluorescein diacetate (H2DCFDA; FanBo), which is commonly used to detect oxidative stress in cells due to the high sensitivity of fluorescence-based assays. After rehydration and incubation for 0 h, 1 h and 4 h at 25 C, the cells were washed and incubated with 20 mM H2DCFDA (dissolved in dimethyl sulfoxide) for 15 min. Cells were prepared and viewed using fluorescence microscopy with filter set number 3 as described. Intracellular Ca2þ overload of freeze-dried M. rouxii sporangiospores was evaluated with Fluo-3-acetoxymethyl ester (Fluo3/AM; Biotium, Hayward, CA, USA). After rehydration and incubation for 0 h, 1 h and 4 h at 25 C, the cells were washed and incubated with 10 mM Fluo-3/AM prepared with a 1 mM stock solution in dimethyl sulfoxide. After washing and resuspension in sterile water, the cells were viewed with a fluorescent microscope using filter set number 3. The rate of nuclear condensation in freeze-dried M. rouxii sporangiospores was evaluated with Hoechst 33258 staining. After rehydration and incubation for 0 h, 6 h and 9 h at 25 C, the cells were washed and stained with Hoechst 33258 as described by Harris et al. [11]. After centrifugation, the cell precipitate was viewed with fluorescence microscopy using filter set number 2 (excitation wavelength 350 nm and emission wavelength 460 nm) for Hoechst 33258. DNA fragmentation by agarose gel electrophoresis was performed. The DNA ladder experiment was performed after rehydration and incubation for 0 h, 12 h, and 24 h. DNA samples for agarose gel electrophoresis were prepared according to the procedure of Cihlar and Sypherd [8] with some modifications. DNA was extracted with CTAB. After treatment with 40 mg/mL RNase A at 37 C for 1 h, the extracts were detected by electrophoresis on 1.5% agarose gel in 1 TAE buffer for 1e1.5 h at 5 V/cm. 2.7. Caspase inhibitor and ROS scavenger studies For metacaspase inhibitor treatment, the fungus was cultivated on PDA for 7e8 d at 28 C and sporangiospores were collected into sterile 10% skimmed milk containing 20 mM Z-VAD-fmk (broadspectrum caspase inhibitor, Promega) and freeze-dried. ROS scavengers, Vc, N-acetyl-cysteine (NAC) and L-proline were used to scavenge ROS and inhibit apoptosis in freeze-dried
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
X. Wang, Y. Wang / Cryobiology xxx (2015) 1e6
M. rouxii. For Vc treatment, 1% Vc was added to the sporangiospore suspension and then the fungus was cultivated on PDA medium for 7e8 d at 28 C and collected. For NAC and L-proline treatment, the fungus was cultivated on PDA containing 20 mM NAC, PDA containing 1.6 mM L-proline, and Czapek-Dox medium containing 1.6 mM L-proline for 7e8 d at 28 C. The sporangiospore suspension and freeze drying were achieved as described above. Then, the rates of nuclear condensation in freeze-dried M. rouxii treated with the caspase inhibitor and ROS scavengers were evaluated by Hoechst 33258 staining as described above. The viability of freeze-dried M. rouxii treated with L-proline was determined with PI staining and the dilution plate method. To further determine whether L-proline has a protective effect on freeze-dried M. rouxii after a long period of preservation, an accelerated storage test (AST) was performed. M. rouxii cultured on PDA medium and C-D medium containing 1.6 mM L-proline was freeze-dried and put in a 37 C water bath for a week. The viability was then tested by the dilution plate method as described.
3. Results 3.1. The viability of freeze-dried M. rouxii After rehydration and incubation, freeze-dried M. rouxii sporangiospores were stained with PI and showed red fluorescence (Fig. 1A). Freeze-dried M. rouxii had a high rate of PI-positivity as well as a delayed peak in PI-positivity (Fig. 1B), while control sporangiospores scarcely showed red fluorescence. The viability using the dilution plate method is 64.89 ± 14.69%, which is much lower than that of the PI test results at 0 h and 12 h, but relatively the same at 24 h possibly because of CIDOCD (cryopreservationinduced delayed-onset cell death) (Fig. 1B).
3
3.2. Apoptotic characteristics in freeze-dried M. rouxii ROS were generated in freeze-dried M. rouxii as determined by intense green fluorescence from H2DCFDA. No cells of the control sporangiospores had ROS-specific fluorescence, while part of the freeze-dried sporangiospores had ROS-positive fluorescence (Fig. 2A). These results demonstrated that ROS were generated in freeze-dried M. rouxii. The rate of ROS generation in freeze-dried M. rouxii was different after rehydration and incubation for different lengths of time (Fig. 2B). The assay using the activated metacaspase CaspACE, FITC-VADfmk In Situ Marker showed that 18.59 ± 2.49% of the freeze-dried sporangiospores following rehydration and 1 h incubation and fluorescence, indicating that metacaspases were activated in contrast to the control cells, where fluorescence was not observed (Fig. 2C and D). There was no fluo-3 positivity in both the freeze-dried group and the control, as detected by the fluorescence of the cellpermeable Ca2þ indicator, fluo-3/AM. On the basis of the apoptosis-inducible factors in freeze-dried M. rouxii, we tested whether there were other apoptotic characteristics. After rehydration and incubation, some of the freeze-dried M. rouxii sporangiospores (Fig. 3) were Hoechst 33258 positive, but none of the control cells fluoresced in response to Hoechst 33258 staining. These results demonstrated that freeze-drying induced nuclear condensation in M. rouxii. Our results indicated the absence of a DNA ladder, which might be due to little or no linker DNA between nucleosomes in M. rouxii. So far among fungal species, only Mucor racemosus has been reported to have a DNA ladder during apoptosis [22]. 3.3. Caspase inhibitor and ROS scavengers study The nuclear condensation rates of freeze-dried M. rouxii treated with caspase inhibitor and ROS scavengers were evaluated with Hoechst 33258 staining. The nuclear condensation rate was significantly decreased in the freeze-dried M. rouxii which was cultured on Czapek-Dox medium containing 1.6 mM L-proline and slightly decreased in the freeze-dried M. rouxii cultured on PDA containing 20 mM NAC. Vc and Z-VAD-fmk showed no effect in decreasing the nuclear condensation rate in freeze-dried M. rouxii (Fig. 4A). The survival rate of freeze-dried M. rouxii cultured on CzapekDox medium containing 1.6 mM L-proline was high in contrast to those cultured on PDA by the dilution plate method. After a week of AST, the survival rate of freeze-dried M. rouxii cultured on CzapekDox medium containing 1.6 mM L-proline was still higher (Fig. 4B). 4. Discussion
Fig. 1. Viability of freeze-dried M. rouxii strain CGMCC 3.2545 as assessed with PI. A. Fluorescence micrograph after staining with PI. B. Mean percentage (±SD) of PI positive cells increased in a time-dependent manner after rehydration and 24 h incubation. Cells were stained with PI, and at least 200 cells were examined with fluorescence microscopy to determine the percentage of dead, fluorescing cells in three independent experiments (two replicates per experiment). Bars ¼ 10 mm.
Apoptosis is a naturally occurring developmental process triggered by various extracellular and intracellular stimuli and has been found to be induced in several fungal species in response to stimuli [7,13]. In this study, apoptosis-associated characteristics such as ROS generation, metacaspase activation, and nuclear condensation occurred in freeze-dried M. rouxii sporangiospores. As far as we know, this is the first study about apoptosis in fungal freeze drying preservation. But apoptosis and the molecular events that occur during cryopreservation have been well studied. The occurrence of apoptosis triggered by cryopreservation was demonstrated in human, bull, boar, and stallion spermatozoa [17,20,23], human bone marrow cells [24], porcine hepatocytes [30], and other types of cells [3]. However, most studies have focused on identifying and quantifying apoptosis following cryopreservation; there are few detailed
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
4
X. Wang, Y. Wang / Cryobiology xxx (2015) 1e6
Fig. 2. Detection of metacaspase activation and ROS generation in freeze-dried M. rouxii. A. Fluorescence micrograph of ROS detection after staining with H2DCFHDA. B. Mean percentage (±SD) of ROS-generating cells. Cells were stained with H2DCFHDA, and at least 200 cells were examined with fluorescence microscopy in three independent experiments (two replicates per experiment). A paired t-test suggested a significant difference. C. Fluorescence micrograph of the assay for metacaspase activation using FITC-VAD-fmk. D. Mean percentage (±SD) of metacaspase activated cells. At least 200 cells were examined with fluorescence microscopy in three independent experiments (two replicates per experiment). Bars ¼ 10 mm.
Fig. 3. A. Fluorescence micrographs of the Hoechst 33258 assay for nuclear condensation in freeze-dried M. rouxii. B. Mean percentage (±SD) of nuclear-condensed cells examined with fluorescence microscopy. Bars ¼ 10 mm.
investigations on the initiating stresses. Inherent in the cryopreservation process is the exposure of cells to numerous stressors, many of which can initiate apoptosis. These stressors include metabolic uncoupling, the production of free radicals, alternations in cell membrane structure and fluidity, dysregulation of cellular ionic balances, release of calcium, and osmotic fluxes [2]. The stresses associated with cryopreservation are by no means complete, and highlight the complexity of the stress response and the multiplicity of potential initiation points. In an effort to provide insight into the effects of the various stressors associated with cryopreservation, studies have begun to focus on the various cellular initiation sites of apoptosis. These studies remain in their infancy, but have begun to lend insight into the pathways associated with cryopreservation-induced molecular cell death, including the cell membrane, nucleus, and mitochondria [3]. In this study, we focused on the following stressors, ROS production, metacaspase
activation, and intracellular Ca2þ overload. Due to the ordered temporal progression of apoptosis cascades, the apoptosis-associated characteristics of freeze-dried M. rouxii were tested after rehydration and incubation for different lengths of time. The generation of ROS is a key mediator of cold-induced apoptosis and contributes to apoptosis during cryopreservation as reported in cultured hepatocytes and liver endothelial cells [10], human spermatozoa and stallion spermatozoa [18,23]. In this study, the results showed that ROS generation in freeze-dried M. rouxii sporangiospores was high immediately after rehydration, suggesting that ROS could be the initiating stress. Caspases are aspartate-specific cysteine proteases that play important roles in mammalian apoptosis. However, caspases have not been identified in fungi. Instead, fungi have a class of related proteases called metacaspases [28]. Caspases have been shown to be activated in
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
X. Wang, Y. Wang / Cryobiology xxx (2015) 1e6
Fig. 4. A. Mean (±SD) percentage of nuclear-condensed cells in freeze-dried M. rouxii treated with the caspase inhibitor and ROS scavengers. A paired t-test suggests there is statistically significant difference between the control (PDA) group and the L-proline group. B. Mean (±SD) percentage survival rate by the dilution plate method. A paired ttest indicates a significant difference between the two groups. M. rouxii cultured in PDA medium and Czapek-Dox medium containing 1.6 mM L-proline was freeze-dried, diluted, and painted on PDA agar. Freeze-dried M. rouxii was put in a 37 C water bath for a week, diluted, and painted on PDA agar.
several cell types after cryopreservation. For example, the activation of caspases 3, 7, and 9 has been detected in stallion spermatozoa [18], the activation of caspase 8 has been detected in humans [19], and the activation of caspase 9 was detected in a bovine model, indicating that caspase activation plays an important role in apoptosis associated with cryopreservation. In this study, the activated metacaspases were detected with ‘CaspACE, FITC-VAD-fmk In Situ Marker, but the broad-spectrum caspase inhibitor Z-VAD-fmk did not decrease the percentage of apoptosis, demonstrating that active metacaspases were likely involved in apoptosis but were not the initial stress in freeze-dried M. rouxii. Intracellular Ca2þ overload or perturbation of intracellular Ca2þ compartmentalization can cause cytotoxicity and trigger either apoptotic or necrotic cell death [6]. Experiments with porcine oocytes as a cell model have shown that cryopreservation leads to increases in the intracellular Ca2þ concentration [16]. In this study, we investigated whether intracellular Ca2þ overload contributes to apoptosis in freeze-dried M. rouxii sporangiospores with Fluo-3/AM. The results showed that there was no Ca2þ overload in freeze-dried M. rouxii. This study also demonstrated that there was nuclear condensation in freeze-dried M. rouxii, suggesting that apoptosis occurred. Moreover, there was a delayed cell death peak (24 h) by PI detection, which was possibly associated with the ordered temporal progression of apoptosis. Apoptosis may result in the inability of the PI assay to differentiate between apoptosis and necrosis, and that may be occurring before the cell membrane integrity is sufficiently affected for PI to detect death [9]. In fact, numerous studies have reported on the molecular-based phenomena of
5
cryopreservation-induced delayed onset cell death. Molecularbased cell death may take many hours to days to manifest following thawing due to the chronological nature of the cell death machinery. It is this temporal component that continues to elude many investigators attempting to characterize molecular cell death following preservation. In 2001, a report detailed the timing of cell death following cryopreservation and termed the phenomena cryopreservation-induced delayed onset cell death (CIDOCD) [4]. Subsequently, a series of investigations on the path of molecular cell death progression ensued. In this study, CIDOCD was also involved in the death of freeze-dried M. rouxii, which further proved that apoptosis contributes to the death of freeze-dried M. rouxii. However, there was no DNA ladder, which might be due to little or no linker DNA between nucleosomes in M. rouxii. Therefore, to determine whether apoptosis occurred in freezedried M. rouxii, further research should be performed. To improve the viability of freeze-dried M. rouxii by inhibiting apoptosis-induced factors, we used a metacaspase inhibitor and ROS scavengers. The caspase inhibitor showed no effect in decreasing the nuclear condensation rate in freeze-dried M. rouxii. Among the ROS scavengers, only L-proline effectively decreased the rate of apoptosis, especially when M. rouxii was grown on CzapekDox medium rather than PDA. This could be due to the multifunction of L-proline, which not only participates in ROS scavenging activity but also acts as a molecular chaperone to protect protein integrity and enhance the activities of different enzymes [27]. Proline treatment can diminish ROS levels in filamentous fungi and yeast, thus preventing programmed cell death [7]. It can also protect human cells against carcinogenic oxidative stress and can reduce lipid peroxidation in alga cells exposed to heavy metals [14]. For a long time, proline was considered an inert compatible osmolyte that protects subcellular structures and macromolecules under osmotic stress [12]. Moreover, proline accumulation can influence stress tolerance in multiple ways and can stabilize ribonucleases and proteases upon arsenate exposure [15]. This is the first study on apoptosis in freeze-dried fungi to demonstrate that apoptosis likely contributes to freeze-drying preservation failure. A union between optimized structural protection and cellemolecular-based modulation is most likely to provide the next level of improvements in post-preservation outcomes. Current cryopreservation studies are now focused on the management of gene-regulated stress-dependent effects on a cell (TAC). Therefore, in the field of freeze drying, we should try to improve viability by inhibiting apoptosis. In summary, this study demonstrates that apoptosis-associated characteristics occur in freeze-dried M. rouxii and that L-proline can decrease the rate of apoptotic characteristics and improve viability. Investigating the effects of freeze drying on cellular biology, such as gene expression, is fundamental for the improvement of techniques and protocols. Future studies will focus on the molecular mechanism and gene expression patterns involved in apoptosis and how L-proline stabilizes cellular homeostasis during freeze-drying conditions and inhibits apoptosis. Conflict of interest There is no conflict of interest. Acknowledgments This study was supported by State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences (08KF031YX2). The authors thank Yuguang Zhou, Head of CGMCC, and all other staff of CGMCC for providing convenience during the research.
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
6
X. Wang, Y. Wang / Cryobiology xxx (2015) 1e6
References [1] J.M. Baust, R.G. Van Buskirk, J.G. Baust, Cryopreservation outcome is enhanced by intracellular-type medium and inhibition of apoptosis, Cryobiology 37 (1998) 410e411. [2] J.M. Baust, R.G. Van Buskirk, J.G. Baust, Gene activation of the apoptotic caspase cascade following cryogenic storage, Cell Preserv. Technol. 1 (2002) 63e80. [3] J.M. Baust, K.K. Snyder, R.G. Van Buskirk, J.G. Baust, Changing paradigms in biopreservation, Biopreserv. Biobank. 7 (2009) 3e12. [4] J.M. Baust, M.J. Vogel, R.G. Van Buskirk, J.G. Baust, A molecular basis of cryopreservation failure and its modulation to improve cell survival, Cell Transpl. 10 (2001) 561e571. [5] A.M. Borman, A. Szekely, C.K. Campbell, E.M. Johnson, Evaluation of the viability of pathogenic filamentous fungi after prolonged storage in sterile water and review of recent published studies on storage methods, Mycopathologia 161 (2006) 361e368. [6] C. Camello-Almaraz, P.J. Gomez-Pinilla, M.J. Pozo, P.J. Camello, Mitochondrial reactive oxygen species and Ca2þ signaling, Am. J. Physiol. Cell Physiol. 291 (2006) 1082e1088. [7] C. Chen, M.B. Dickman, Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 3459e3464. [8] R.L. Cihlar, P.S. Sypherd, The organization of the ribosomal RNA genes in the fungus Mucor racemosus, Nucleic Acids Res. 8 (1980) 793e804. [9] L.M. Cosentino, W. Corwin, J.M. Baust, N. Diaz-Mayoral, H. Cooley, W. Shao, R. Van Buskirk, J.G. Baust, Preliminary report: evaluation of storage conditions and cryococktails during peripheral blood mononuclear cell cryopreservation, Cell Preserv. Technol. 5 (2007) 189e204. [10] R. Fujita, T. Hui, M. Chelly, A.A. Demetriou, The effect of antioxidants and a caspase inhibitor on cryopreserved rat hepatocytes, Cell Transpl. 14 (2005) 391e396. [11] S.D. Harris, J.L. Morrell, J.E. Hamer, Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis, Genetics 136 (1994) 517e532. [12] P.K. Kishor, S. Sangam, R. Amrutha, P.S. Laxmi, K. Naidu, K. Rao, S. Rao, K. Reddy, P. Theriappan, N. Sreenivasulu, Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: its implications in plant growth and abiotic stress tolerance, Curr. Sci. 88 (2005) 424e438. [13] S.M. Marek, J. Wu, N.L. Glass, D.G. Gilchrist, R.M. Bostock, Nuclear DNA degradation during heterokaryon incompatibility in Neurospora crassa, Fungal Genet. Biol. 40 (2003) 126e137. [14] S. Mehta, J. Gaur, Heavy-metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris, New Phytol. 143 (1999) 253e259. [15] S. Mishra, R.S. Dubey, Inhibition of ribonuclease and protease activities in arsenic exposed rice seedlings: role of proline as enzyme protectant, J. Plant Physiol. 163 (2006) 927e936. [16] S. Nakagawa, A. Yoneda, K. Hayakawa, T. Watanabe, Improvement in the
[17]
[18]
[19]
[20]
[21]
[22] [23] [24]
[25] [26]
[27] [28]
[29]
[30]
in vitro maturation rate of porcine oocytes vitrified at the germinal vesicle stage by treatment with a mitochondrial permeability transition inhibitor, Cryobiology 57 (2008) 269e275. C. Ortega-Ferrusola, L. Gonz alez-Fern andez, J.M. Morrell, C. Salazar-Sandoval, ~ a, Lipid peroxidaB. Macías-García, H. Rodríguez-Martinez, J.A. Tapia, F.J. Pen tion, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is related to apoptotic-like changes, Reproduction 138 (2009) 55e63. n, E. Varela-Fern C. Ortega-Ferrusola, Y. Sotillo-Gala andez, J.M. Gallardo~ os, A. Muriel, L. Gonza lez-Fern Bolan andez, J.A. Tapia, F.J. Pena, Detection of “apoptosis-like” changes during the cryopreservation process in equine sperm, J. Androl. 29 (2008) 213e221. U. Paasch, R.K. Sharma, A.K. Gupta, S. Grunewald, E.J. Mascha, A.J. Thomas, H.J. Glander, A. Agarwal, Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa, Biol. Reprod. 71 (2004) 1828e1837. ~ a, A. Johannisson, M. Wallgren, H.R. Martinez, Antioxidant supplemenF. Pen tation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture, Zygote 12 (2004) 117e124. E. Pinto, L. Vale-Silva, C. Cavaleiro, L. Salgueiro, Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species, J. Med. Microbiol. 58 (2009) 1454e1462. L. Roze, J. Linz, Lovastatin triggers an apoptosis-like cell death process in the fungus Mucor racemosus, Fungal Genet. Biol. 25 (1998) 119e133. T.M. Said, A. Gaglani, A. Agarwal, Implication of apoptosis in sperm cryoinjury, Reprod. Biomed. Online 21 (2010) 456e462. €m-Lindberg, B. Joseph, B. Zhivotovsky, Freezing J. Schmidt-Mende, E. Hellstro induces artificial cleavage of apoptosis-related proteins in human bone marrow cells, J. Immunol. Methods 245 (2000) 91e94. D. Smith, Long-term preservation of test strains (fungus), Int. Biodeter. Biodegr. 31 (1993) 227e230. S.K. Sundari, A. Adholeya, Freeze-drying vegetative mycelium of Laccaria fraterna and its subsequent regeneration, Biotechnol. Tech. 13 (1999) 491e495. L. Szabados, A. Savoure, Proline: a multifunctional amino acid, Trends Plant Sci. 15 (2010) 89e97. A.G. Uren, K. O'Rourke, L. Aravind, M.T. Pisabarro, S. Seshagiri, E.V. Koonin, V.M. Dixit, Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma, Mol. Cell 6 (2000) 961e967. X.-Z. Wu, W.-Q. Chang, A.-X. Cheng, L.-M. Sun, H.-X. Lou, Plagiochin E, an antifungal active macrocyclic bis (bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway, BBA-Gen. Subj. 1800 (2010) 439e447. T. Yagi, J.A. Hardin, Y.M. Valenzuela, H. Miyoshi, G.J. Gores, S.L. Nyberg, Caspase inhibition reduces apoptotic death of cryopreserved porcine hepatocytes, Hepatology 33 (2001) 1432e1440.
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline Xiaoyun Wang, Youzhi Wang* China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 September 2015 Received in revised form 20 November 2015 Accepted 30 November 2015 Available online xxx
Freeze-drying is one of the most effective methods to preserve fungi for an extended period. However, it is associated with a loss of viability and shortened storage time in some fungi. This study evaluated the stresses that led to the death of freeze-dried Mucor rouxii by using cell apoptotic methods. The results showed there were apoptosis-inducing stresses, such as the generation of obvious intracellular reactive oxygen species (ROS) and metacaspase activation. Moreover, nuclear condensation and a delayed cell death peak were determined after rehydration and 24 h incubation in freeze-dried M. rouxii via a propidium iodide (PI) assay, which is similar to the phenomenon of cryopreservation-induced delayed-onset cell death (CIDOCD). Then, several protective agents were tested to decrease the apoptosis-inducing stresses and to improve the viability. Finally, it was found that 1.6 mM L-proline can effectively decrease the nuclear condensation rate and increase the survival rate in freeze-dried M. rouxii. In conclusion: (1) apoptosis-inducing factors occur in freeze-dried M. rouxii. (2) ROS and activated metacaspases lead to death in freeze-dried M. rouxii. (3) L-proline increases the survival rate of freeze-dried M. rouxii. © 2015 Elsevier Inc. All rights reserved.
Keywords: Apoptosis Cryopreservation Freeze-drying L-proline Viability Mucor rouxii
1. Introduction Freeze drying is a convenient technique favored by many large culture collections. Freeze-drying of an isolate can be performed by freezing and drying a suspension of the fungus in its nutritive medium using a freeze dryer in conjunction with a vacuum apparatus. Ampoules with freeze-dried fungi are sealed to prevent damage of lyophilized material as a result of contact with air. With this technique, it is common for the fungal conidia to survive [5,25]. Moreover, storage by freeze-drying is one of the most suitable methods for maintaining viable and stable fungal cultures for 20e40 years. It is a convenient and successful method for preserving sporulating fungi and is ideal for the distribution of the organisms. However, there are some restrictions, such as loss of viability and a shortened storage time in some fungi. Optimization of the freeze-drying protocol included selection of the candidate fungus, optimizing physiological growth conditions and age, standardizing the protectant type and concentration, optimizing the pre-freezing
* Corresponding author. E-mail address: [email protected] (Y. Wang).
method, freeze-drying run and extent of drying, and choosing the rehydrant and the extent of rehydration [26]. As far as we know, this is the first study on apoptosis in the field of freeze-driedpreserved fungi. The field of cryopreservation has a long and successful history of in depth study and progress. In 1998, several independent groups reported the direct involvement of apoptotic and necrotic cell death following cryopreservation [1]. Many studies have identified diverse molecular-based cellular responses to cryopreservation and have further demonstrated significant improvements in cell survival through the inhibition of apoptosis. A substantial volume of studies have described the modulation of cell death through the use of various protease inhibitors, free radical scavengers, media formulations, and other novel compounds [1,3]. However, understanding of the pathway activation, progression, control and the downstream effect on cell apoptosis during cryopreservation remains in its infancy. Modulation studies, such as targeted apoptotic control (TAC), have shown promise in furthering our understanding of the activation pathways and are proving to be a critical next step in the evolution of cryopreservation sciences [3]). Therefore, the aims of this study were to answer the following questions about freeze-dried Mucor rouxii: 1) Are apoptosis-
http://dx.doi.org/10.1016/j.cryobiol.2015.11.006 0011-2240/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
2
X. Wang, Y. Wang / Cryobiology xxx (2015) 1e6
inducing factors involved in the death of freeze-dried M. rouxii? 2) Do apoptosis inhibitors such as caspase inhibitor ZVAD-fmk and ROS-scavengers have a protective effect on freeze-dried M. rouxii? These questions have general importance to the understanding of fungal death during freeze-drying and special importance for the use of freeze-dried M. rouxii in many applications. 2. Materials and methods 2.1. Strain and culture conditions M. rouxii strain CGMCC 3.2545, from the China General Microbiological Culture Collection Center, was used in this study. The fungus was cultivated on PDA (20% potato extract, 2% dextrose, 2% agar) medium or Czapek-Dox medium (0.2% sodium nitrate, 0.1% potassium phosphate dibasic, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferric sulfate, 3% sucrose) for 7e8 d at 28 C. 2.2. Freeze-drying of M. rouxii Fungal cultures at 7 days old were selected for freeze drying according to the standard procedure in CGMCC. Approximately 4 ml of sterile skimmed milk was added to a slanted culture and the surface of the agar was gently scraped with a sterile blade to obtain a sporangiospore suspension, which was filtered through four layers of sterile lens wiping paper to remove hyphal fragments. The sporangiospore suspension was added to each ampoule which was then loosely plugged with a cotton swab and put in a 20 C refrigerator for pre-freezing. Sporangiospores were freeze-dried and then sealed under vacuum using a gas-oxygen torch and stored at 4 C. At least three ampoules were used in each of the following experiments. Freeze-dried M. rouxii sporangiospores (106 sporangiospores/mL) were rehydrated with sterile water and used for the following tests; the sporangiospores before pre-freezing were used as a control. 2.3. Viability assay Dead or dying cells with disrupted membranes were detected microscopically using propidium iodide (PI), a nucleic-acid-binding fluorescent probe that is excluded by intact membranes of viable cells, as well as the procedure of Pinto et al. [21] with some modifications. After rehydration and incubation for 0 h, 12 h and 24 h at 25 C, cells were washed (2000 g for 3 min) and stained with 1 mg/ mL PI (Sigma, St. Louis, Missouri) for 5 min. After centrifugation, a sample of the cell precipitate for each treatment was viewed on a glass slide with a ZEISS Axioplan 2 fluorescent microscope using filter set 4 for PI (excitation wavelength 488 nm and emission wavelength 615 nm). The percentage of viable cells was determined as: [viable cells/(dead cells þ viable cells)] 100%. The dilution plate method was also used to test the survival rate of freeze-dried M. rouxii sporangiospores. Before pre-freezing, 0.2 ml M. rouxii spore suspension was diluted 104 times, and 0.1 ml was painted on PDA plates. After culturing in a 25 C incubator for approximately 17e19 h, colony-forming units (CFUs) were counted as NA. After freeze drying, M. rouxii sporangiospore powder was rehydrated, diluted, and painted on PDA plates as described. The CFUs were counted as NB. The survival rate was calculated by NB/NA 100%. 2.4. Fluorescence and light microscopy For each replicate of each treatment, at least 200 cells were examined with a ZEISS Axioplan 2 microscope (Germany) for
fluorescence applications and filter set 5 for light microscopy with differential interference contrast. All images were captured with an AxioCamMR5 camera and processed by Adobe PhotoShop 7.0. 2.5. Statistical analyses All tests were performed at least in triplicate. The results are expressed as the mean ± SD (standard deviation). The comparison between the controls and the freeze-dried M. rouxii that was treated with the caspase inhibitor and ROS scavengers was analyzed with a paired t-test using SPSS17.0. P < 0.05 was considered to indicate a statistically significant difference. 2.6. Apoptosis analysis Activated metacaspases were detected microscopically with CaspACE FITC-VAD-fmk In Situ Marker (Promega, Madison, USA) as described by Wu et al. [29]. After rehydration and incubation for 0 h, 1 h and 4 h at 25 C, the cells were washed and stained with FITC-VAD-FMK according to the manufacturer's instructions. Cells were washed and precipitated as above for fluorescent microscopy using filter set number 3 (excitation wavelength 488 nm and emission wavelength 530 nm). The intracellular production of reactive oxygen species (ROS) was detected with the oxidant-sensitive probe dichlorodihydrofluorescein diacetate (H2DCFDA; FanBo), which is commonly used to detect oxidative stress in cells due to the high sensitivity of fluorescence-based assays. After rehydration and incubation for 0 h, 1 h and 4 h at 25 C, the cells were washed and incubated with 20 mM H2DCFDA (dissolved in dimethyl sulfoxide) for 15 min. Cells were prepared and viewed using fluorescence microscopy with filter set number 3 as described. Intracellular Ca2þ overload of freeze-dried M. rouxii sporangiospores was evaluated with Fluo-3-acetoxymethyl ester (Fluo3/AM; Biotium, Hayward, CA, USA). After rehydration and incubation for 0 h, 1 h and 4 h at 25 C, the cells were washed and incubated with 10 mM Fluo-3/AM prepared with a 1 mM stock solution in dimethyl sulfoxide. After washing and resuspension in sterile water, the cells were viewed with a fluorescent microscope using filter set number 3. The rate of nuclear condensation in freeze-dried M. rouxii sporangiospores was evaluated with Hoechst 33258 staining. After rehydration and incubation for 0 h, 6 h and 9 h at 25 C, the cells were washed and stained with Hoechst 33258 as described by Harris et al. [11]. After centrifugation, the cell precipitate was viewed with fluorescence microscopy using filter set number 2 (excitation wavelength 350 nm and emission wavelength 460 nm) for Hoechst 33258. DNA fragmentation by agarose gel electrophoresis was performed. The DNA ladder experiment was performed after rehydration and incubation for 0 h, 12 h, and 24 h. DNA samples for agarose gel electrophoresis were prepared according to the procedure of Cihlar and Sypherd [8] with some modifications. DNA was extracted with CTAB. After treatment with 40 mg/mL RNase A at 37 C for 1 h, the extracts were detected by electrophoresis on 1.5% agarose gel in 1 TAE buffer for 1e1.5 h at 5 V/cm. 2.7. Caspase inhibitor and ROS scavenger studies For metacaspase inhibitor treatment, the fungus was cultivated on PDA for 7e8 d at 28 C and sporangiospores were collected into sterile 10% skimmed milk containing 20 mM Z-VAD-fmk (broadspectrum caspase inhibitor, Promega) and freeze-dried. ROS scavengers, Vc, N-acetyl-cysteine (NAC) and L-proline were used to scavenge ROS and inhibit apoptosis in freeze-dried
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
X. Wang, Y. Wang / Cryobiology xxx (2015) 1e6
M. rouxii. For Vc treatment, 1% Vc was added to the sporangiospore suspension and then the fungus was cultivated on PDA medium for 7e8 d at 28 C and collected. For NAC and L-proline treatment, the fungus was cultivated on PDA containing 20 mM NAC, PDA containing 1.6 mM L-proline, and Czapek-Dox medium containing 1.6 mM L-proline for 7e8 d at 28 C. The sporangiospore suspension and freeze drying were achieved as described above. Then, the rates of nuclear condensation in freeze-dried M. rouxii treated with the caspase inhibitor and ROS scavengers were evaluated by Hoechst 33258 staining as described above. The viability of freeze-dried M. rouxii treated with L-proline was determined with PI staining and the dilution plate method. To further determine whether L-proline has a protective effect on freeze-dried M. rouxii after a long period of preservation, an accelerated storage test (AST) was performed. M. rouxii cultured on PDA medium and C-D medium containing 1.6 mM L-proline was freeze-dried and put in a 37 C water bath for a week. The viability was then tested by the dilution plate method as described.
3. Results 3.1. The viability of freeze-dried M. rouxii After rehydration and incubation, freeze-dried M. rouxii sporangiospores were stained with PI and showed red fluorescence (Fig. 1A). Freeze-dried M. rouxii had a high rate of PI-positivity as well as a delayed peak in PI-positivity (Fig. 1B), while control sporangiospores scarcely showed red fluorescence. The viability using the dilution plate method is 64.89 ± 14.69%, which is much lower than that of the PI test results at 0 h and 12 h, but relatively the same at 24 h possibly because of CIDOCD (cryopreservationinduced delayed-onset cell death) (Fig. 1B).
3
3.2. Apoptotic characteristics in freeze-dried M. rouxii ROS were generated in freeze-dried M. rouxii as determined by intense green fluorescence from H2DCFDA. No cells of the control sporangiospores had ROS-specific fluorescence, while part of the freeze-dried sporangiospores had ROS-positive fluorescence (Fig. 2A). These results demonstrated that ROS were generated in freeze-dried M. rouxii. The rate of ROS generation in freeze-dried M. rouxii was different after rehydration and incubation for different lengths of time (Fig. 2B). The assay using the activated metacaspase CaspACE, FITC-VADfmk In Situ Marker showed that 18.59 ± 2.49% of the freeze-dried sporangiospores following rehydration and 1 h incubation and fluorescence, indicating that metacaspases were activated in contrast to the control cells, where fluorescence was not observed (Fig. 2C and D). There was no fluo-3 positivity in both the freeze-dried group and the control, as detected by the fluorescence of the cellpermeable Ca2þ indicator, fluo-3/AM. On the basis of the apoptosis-inducible factors in freeze-dried M. rouxii, we tested whether there were other apoptotic characteristics. After rehydration and incubation, some of the freeze-dried M. rouxii sporangiospores (Fig. 3) were Hoechst 33258 positive, but none of the control cells fluoresced in response to Hoechst 33258 staining. These results demonstrated that freeze-drying induced nuclear condensation in M. rouxii. Our results indicated the absence of a DNA ladder, which might be due to little or no linker DNA between nucleosomes in M. rouxii. So far among fungal species, only Mucor racemosus has been reported to have a DNA ladder during apoptosis [22]. 3.3. Caspase inhibitor and ROS scavengers study The nuclear condensation rates of freeze-dried M. rouxii treated with caspase inhibitor and ROS scavengers were evaluated with Hoechst 33258 staining. The nuclear condensation rate was significantly decreased in the freeze-dried M. rouxii which was cultured on Czapek-Dox medium containing 1.6 mM L-proline and slightly decreased in the freeze-dried M. rouxii cultured on PDA containing 20 mM NAC. Vc and Z-VAD-fmk showed no effect in decreasing the nuclear condensation rate in freeze-dried M. rouxii (Fig. 4A). The survival rate of freeze-dried M. rouxii cultured on CzapekDox medium containing 1.6 mM L-proline was high in contrast to those cultured on PDA by the dilution plate method. After a week of AST, the survival rate of freeze-dried M. rouxii cultured on CzapekDox medium containing 1.6 mM L-proline was still higher (Fig. 4B). 4. Discussion
Fig. 1. Viability of freeze-dried M. rouxii strain CGMCC 3.2545 as assessed with PI. A. Fluorescence micrograph after staining with PI. B. Mean percentage (±SD) of PI positive cells increased in a time-dependent manner after rehydration and 24 h incubation. Cells were stained with PI, and at least 200 cells were examined with fluorescence microscopy to determine the percentage of dead, fluorescing cells in three independent experiments (two replicates per experiment). Bars ¼ 10 mm.
Apoptosis is a naturally occurring developmental process triggered by various extracellular and intracellular stimuli and has been found to be induced in several fungal species in response to stimuli [7,13]. In this study, apoptosis-associated characteristics such as ROS generation, metacaspase activation, and nuclear condensation occurred in freeze-dried M. rouxii sporangiospores. As far as we know, this is the first study about apoptosis in fungal freeze drying preservation. But apoptosis and the molecular events that occur during cryopreservation have been well studied. The occurrence of apoptosis triggered by cryopreservation was demonstrated in human, bull, boar, and stallion spermatozoa [17,20,23], human bone marrow cells [24], porcine hepatocytes [30], and other types of cells [3]. However, most studies have focused on identifying and quantifying apoptosis following cryopreservation; there are few detailed
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
4
X. Wang, Y. Wang / Cryobiology xxx (2015) 1e6
Fig. 2. Detection of metacaspase activation and ROS generation in freeze-dried M. rouxii. A. Fluorescence micrograph of ROS detection after staining with H2DCFHDA. B. Mean percentage (±SD) of ROS-generating cells. Cells were stained with H2DCFHDA, and at least 200 cells were examined with fluorescence microscopy in three independent experiments (two replicates per experiment). A paired t-test suggested a significant difference. C. Fluorescence micrograph of the assay for metacaspase activation using FITC-VAD-fmk. D. Mean percentage (±SD) of metacaspase activated cells. At least 200 cells were examined with fluorescence microscopy in three independent experiments (two replicates per experiment). Bars ¼ 10 mm.
Fig. 3. A. Fluorescence micrographs of the Hoechst 33258 assay for nuclear condensation in freeze-dried M. rouxii. B. Mean percentage (±SD) of nuclear-condensed cells examined with fluorescence microscopy. Bars ¼ 10 mm.
investigations on the initiating stresses. Inherent in the cryopreservation process is the exposure of cells to numerous stressors, many of which can initiate apoptosis. These stressors include metabolic uncoupling, the production of free radicals, alternations in cell membrane structure and fluidity, dysregulation of cellular ionic balances, release of calcium, and osmotic fluxes [2]. The stresses associated with cryopreservation are by no means complete, and highlight the complexity of the stress response and the multiplicity of potential initiation points. In an effort to provide insight into the effects of the various stressors associated with cryopreservation, studies have begun to focus on the various cellular initiation sites of apoptosis. These studies remain in their infancy, but have begun to lend insight into the pathways associated with cryopreservation-induced molecular cell death, including the cell membrane, nucleus, and mitochondria [3]. In this study, we focused on the following stressors, ROS production, metacaspase
activation, and intracellular Ca2þ overload. Due to the ordered temporal progression of apoptosis cascades, the apoptosis-associated characteristics of freeze-dried M. rouxii were tested after rehydration and incubation for different lengths of time. The generation of ROS is a key mediator of cold-induced apoptosis and contributes to apoptosis during cryopreservation as reported in cultured hepatocytes and liver endothelial cells [10], human spermatozoa and stallion spermatozoa [18,23]. In this study, the results showed that ROS generation in freeze-dried M. rouxii sporangiospores was high immediately after rehydration, suggesting that ROS could be the initiating stress. Caspases are aspartate-specific cysteine proteases that play important roles in mammalian apoptosis. However, caspases have not been identified in fungi. Instead, fungi have a class of related proteases called metacaspases [28]. Caspases have been shown to be activated in
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
X. Wang, Y. Wang / Cryobiology xxx (2015) 1e6
Fig. 4. A. Mean (±SD) percentage of nuclear-condensed cells in freeze-dried M. rouxii treated with the caspase inhibitor and ROS scavengers. A paired t-test suggests there is statistically significant difference between the control (PDA) group and the L-proline group. B. Mean (±SD) percentage survival rate by the dilution plate method. A paired ttest indicates a significant difference between the two groups. M. rouxii cultured in PDA medium and Czapek-Dox medium containing 1.6 mM L-proline was freeze-dried, diluted, and painted on PDA agar. Freeze-dried M. rouxii was put in a 37 C water bath for a week, diluted, and painted on PDA agar.
several cell types after cryopreservation. For example, the activation of caspases 3, 7, and 9 has been detected in stallion spermatozoa [18], the activation of caspase 8 has been detected in humans [19], and the activation of caspase 9 was detected in a bovine model, indicating that caspase activation plays an important role in apoptosis associated with cryopreservation. In this study, the activated metacaspases were detected with ‘CaspACE, FITC-VAD-fmk In Situ Marker, but the broad-spectrum caspase inhibitor Z-VAD-fmk did not decrease the percentage of apoptosis, demonstrating that active metacaspases were likely involved in apoptosis but were not the initial stress in freeze-dried M. rouxii. Intracellular Ca2þ overload or perturbation of intracellular Ca2þ compartmentalization can cause cytotoxicity and trigger either apoptotic or necrotic cell death [6]. Experiments with porcine oocytes as a cell model have shown that cryopreservation leads to increases in the intracellular Ca2þ concentration [16]. In this study, we investigated whether intracellular Ca2þ overload contributes to apoptosis in freeze-dried M. rouxii sporangiospores with Fluo-3/AM. The results showed that there was no Ca2þ overload in freeze-dried M. rouxii. This study also demonstrated that there was nuclear condensation in freeze-dried M. rouxii, suggesting that apoptosis occurred. Moreover, there was a delayed cell death peak (24 h) by PI detection, which was possibly associated with the ordered temporal progression of apoptosis. Apoptosis may result in the inability of the PI assay to differentiate between apoptosis and necrosis, and that may be occurring before the cell membrane integrity is sufficiently affected for PI to detect death [9]. In fact, numerous studies have reported on the molecular-based phenomena of
5
cryopreservation-induced delayed onset cell death. Molecularbased cell death may take many hours to days to manifest following thawing due to the chronological nature of the cell death machinery. It is this temporal component that continues to elude many investigators attempting to characterize molecular cell death following preservation. In 2001, a report detailed the timing of cell death following cryopreservation and termed the phenomena cryopreservation-induced delayed onset cell death (CIDOCD) [4]. Subsequently, a series of investigations on the path of molecular cell death progression ensued. In this study, CIDOCD was also involved in the death of freeze-dried M. rouxii, which further proved that apoptosis contributes to the death of freeze-dried M. rouxii. However, there was no DNA ladder, which might be due to little or no linker DNA between nucleosomes in M. rouxii. Therefore, to determine whether apoptosis occurred in freezedried M. rouxii, further research should be performed. To improve the viability of freeze-dried M. rouxii by inhibiting apoptosis-induced factors, we used a metacaspase inhibitor and ROS scavengers. The caspase inhibitor showed no effect in decreasing the nuclear condensation rate in freeze-dried M. rouxii. Among the ROS scavengers, only L-proline effectively decreased the rate of apoptosis, especially when M. rouxii was grown on CzapekDox medium rather than PDA. This could be due to the multifunction of L-proline, which not only participates in ROS scavenging activity but also acts as a molecular chaperone to protect protein integrity and enhance the activities of different enzymes [27]. Proline treatment can diminish ROS levels in filamentous fungi and yeast, thus preventing programmed cell death [7]. It can also protect human cells against carcinogenic oxidative stress and can reduce lipid peroxidation in alga cells exposed to heavy metals [14]. For a long time, proline was considered an inert compatible osmolyte that protects subcellular structures and macromolecules under osmotic stress [12]. Moreover, proline accumulation can influence stress tolerance in multiple ways and can stabilize ribonucleases and proteases upon arsenate exposure [15]. This is the first study on apoptosis in freeze-dried fungi to demonstrate that apoptosis likely contributes to freeze-drying preservation failure. A union between optimized structural protection and cellemolecular-based modulation is most likely to provide the next level of improvements in post-preservation outcomes. Current cryopreservation studies are now focused on the management of gene-regulated stress-dependent effects on a cell (TAC). Therefore, in the field of freeze drying, we should try to improve viability by inhibiting apoptosis. In summary, this study demonstrates that apoptosis-associated characteristics occur in freeze-dried M. rouxii and that L-proline can decrease the rate of apoptotic characteristics and improve viability. Investigating the effects of freeze drying on cellular biology, such as gene expression, is fundamental for the improvement of techniques and protocols. Future studies will focus on the molecular mechanism and gene expression patterns involved in apoptosis and how L-proline stabilizes cellular homeostasis during freeze-drying conditions and inhibits apoptosis. Conflict of interest There is no conflict of interest. Acknowledgments This study was supported by State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences (08KF031YX2). The authors thank Yuguang Zhou, Head of CGMCC, and all other staff of CGMCC for providing convenience during the research.
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006
6
X. Wang, Y. Wang / Cryobiology xxx (2015) 1e6
References [1] J.M. Baust, R.G. Van Buskirk, J.G. Baust, Cryopreservation outcome is enhanced by intracellular-type medium and inhibition of apoptosis, Cryobiology 37 (1998) 410e411. [2] J.M. Baust, R.G. Van Buskirk, J.G. Baust, Gene activation of the apoptotic caspase cascade following cryogenic storage, Cell Preserv. Technol. 1 (2002) 63e80. [3] J.M. Baust, K.K. Snyder, R.G. Van Buskirk, J.G. Baust, Changing paradigms in biopreservation, Biopreserv. Biobank. 7 (2009) 3e12. [4] J.M. Baust, M.J. Vogel, R.G. Van Buskirk, J.G. Baust, A molecular basis of cryopreservation failure and its modulation to improve cell survival, Cell Transpl. 10 (2001) 561e571. [5] A.M. Borman, A. Szekely, C.K. Campbell, E.M. Johnson, Evaluation of the viability of pathogenic filamentous fungi after prolonged storage in sterile water and review of recent published studies on storage methods, Mycopathologia 161 (2006) 361e368. [6] C. Camello-Almaraz, P.J. Gomez-Pinilla, M.J. Pozo, P.J. Camello, Mitochondrial reactive oxygen species and Ca2þ signaling, Am. J. Physiol. Cell Physiol. 291 (2006) 1082e1088. [7] C. Chen, M.B. Dickman, Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 3459e3464. [8] R.L. Cihlar, P.S. Sypherd, The organization of the ribosomal RNA genes in the fungus Mucor racemosus, Nucleic Acids Res. 8 (1980) 793e804. [9] L.M. Cosentino, W. Corwin, J.M. Baust, N. Diaz-Mayoral, H. Cooley, W. Shao, R. Van Buskirk, J.G. Baust, Preliminary report: evaluation of storage conditions and cryococktails during peripheral blood mononuclear cell cryopreservation, Cell Preserv. Technol. 5 (2007) 189e204. [10] R. Fujita, T. Hui, M. Chelly, A.A. Demetriou, The effect of antioxidants and a caspase inhibitor on cryopreserved rat hepatocytes, Cell Transpl. 14 (2005) 391e396. [11] S.D. Harris, J.L. Morrell, J.E. Hamer, Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis, Genetics 136 (1994) 517e532. [12] P.K. Kishor, S. Sangam, R. Amrutha, P.S. Laxmi, K. Naidu, K. Rao, S. Rao, K. Reddy, P. Theriappan, N. Sreenivasulu, Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: its implications in plant growth and abiotic stress tolerance, Curr. Sci. 88 (2005) 424e438. [13] S.M. Marek, J. Wu, N.L. Glass, D.G. Gilchrist, R.M. Bostock, Nuclear DNA degradation during heterokaryon incompatibility in Neurospora crassa, Fungal Genet. Biol. 40 (2003) 126e137. [14] S. Mehta, J. Gaur, Heavy-metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris, New Phytol. 143 (1999) 253e259. [15] S. Mishra, R.S. Dubey, Inhibition of ribonuclease and protease activities in arsenic exposed rice seedlings: role of proline as enzyme protectant, J. Plant Physiol. 163 (2006) 927e936. [16] S. Nakagawa, A. Yoneda, K. Hayakawa, T. Watanabe, Improvement in the
[17]
[18]
[19]
[20]
[21]
[22] [23] [24]
[25] [26]
[27] [28]
[29]
[30]
in vitro maturation rate of porcine oocytes vitrified at the germinal vesicle stage by treatment with a mitochondrial permeability transition inhibitor, Cryobiology 57 (2008) 269e275. C. Ortega-Ferrusola, L. Gonz alez-Fern andez, J.M. Morrell, C. Salazar-Sandoval, ~ a, Lipid peroxidaB. Macías-García, H. Rodríguez-Martinez, J.A. Tapia, F.J. Pen tion, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is related to apoptotic-like changes, Reproduction 138 (2009) 55e63. n, E. Varela-Fern C. Ortega-Ferrusola, Y. Sotillo-Gala andez, J.M. Gallardo~ os, A. Muriel, L. Gonza lez-Fern Bolan andez, J.A. Tapia, F.J. Pena, Detection of “apoptosis-like” changes during the cryopreservation process in equine sperm, J. Androl. 29 (2008) 213e221. U. Paasch, R.K. Sharma, A.K. Gupta, S. Grunewald, E.J. Mascha, A.J. Thomas, H.J. Glander, A. Agarwal, Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa, Biol. Reprod. 71 (2004) 1828e1837. ~ a, A. Johannisson, M. Wallgren, H.R. Martinez, Antioxidant supplemenF. Pen tation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture, Zygote 12 (2004) 117e124. E. Pinto, L. Vale-Silva, C. Cavaleiro, L. Salgueiro, Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species, J. Med. Microbiol. 58 (2009) 1454e1462. L. Roze, J. Linz, Lovastatin triggers an apoptosis-like cell death process in the fungus Mucor racemosus, Fungal Genet. Biol. 25 (1998) 119e133. T.M. Said, A. Gaglani, A. Agarwal, Implication of apoptosis in sperm cryoinjury, Reprod. Biomed. Online 21 (2010) 456e462. €m-Lindberg, B. Joseph, B. Zhivotovsky, Freezing J. Schmidt-Mende, E. Hellstro induces artificial cleavage of apoptosis-related proteins in human bone marrow cells, J. Immunol. Methods 245 (2000) 91e94. D. Smith, Long-term preservation of test strains (fungus), Int. Biodeter. Biodegr. 31 (1993) 227e230. S.K. Sundari, A. Adholeya, Freeze-drying vegetative mycelium of Laccaria fraterna and its subsequent regeneration, Biotechnol. Tech. 13 (1999) 491e495. L. Szabados, A. Savoure, Proline: a multifunctional amino acid, Trends Plant Sci. 15 (2010) 89e97. A.G. Uren, K. O'Rourke, L. Aravind, M.T. Pisabarro, S. Seshagiri, E.V. Koonin, V.M. Dixit, Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma, Mol. Cell 6 (2000) 961e967. X.-Z. Wu, W.-Q. Chang, A.-X. Cheng, L.-M. Sun, H.-X. Lou, Plagiochin E, an antifungal active macrocyclic bis (bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway, BBA-Gen. Subj. 1800 (2010) 439e447. T. Yagi, J.A. Hardin, Y.M. Valenzuela, H. Miyoshi, G.J. Gores, S.L. Nyberg, Caspase inhibition reduces apoptotic death of cryopreserved porcine hepatocytes, Hepatology 33 (2001) 1432e1440.
Please cite this article in press as: X. Wang, Y. Wang, Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.006