Cryobiology xxx (2015) xxx–xxx
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Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming Jakkhaphan Chasombat a, Takashi Nagai b,c, Rangsun Parnpai d, Thevin Vongpralub a,⇑ a
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand Food and Fertilizer Technology Center, Taipei 10648, Taiwan NARO Institute of Livestock and Grassland Science, Tsukuba, Japan d Embryo Technology and Stem Cell Research Center and School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand b c
a r t i c l e
i n f o
Article history: Received 28 March 2015 Revised 16 June 2015 Accepted 9 July 2015 Available online xxxx Keywords: Docetaxel Bovine oocytes Vitrification Cytoskeleton fibers
a b s t r a c t The stabilization of spindle fibersis important for successful vitrification of bovine oocytes because microtubules and other cytoskeleton fibers (CSF) can be damaged during vitrification, resulting in failure of fertilization after thawing. Docetaxel, a stabilizing agent, could potentially reduce CSF damage of bovine oocytes induced during vitrification. However, there have been no reports on the effects of docetaxel on their vitrification. Experiment 1 was conducted to investigate the effects of various doses of docetaxel (0.0, 0.05, 0.5, 5.0 and 50 lM) in preincubation medium of in vitro matured (IVM) bovine oocytes on their developmental ability after in vitro fertilization (IVF). The results show that 0.05 lM docetaxel had no adverse effect on embryo development, while docetaxel at a concentration of P0.5 lM inhibited development. Experiments 2 and 3 were conducted to investigate the effects of preincubation of IVM bovine oocytes with 0.05 lM docetaxel for 30 min prior to vitrification-warming on CSF integrity (Experiment 2), and on oocyte survival and viability after IVF (Experiment 3). When preincubated with 0.05 lM docetaxel for 30 min before vitrification, post-thawed oocytes had less CSF damage and higher survival rates compared with those untreated with docetaxel before vitrification. Surviving oocytes also had higher rates of cleavage and development to the blastocyst stage after IVF. In conclusion, preincubation of IVM bovine oocytes with 0.05 lM docetaxel for 30 min before vitrification was effective at preventing CSF damage during vitrification, and improving oocyte viability after warming and subsequent cleavage and blastocyst formation after IVF. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Vitrification of oocytes is a powerful method for preservation of genetic diversity in endangered animal species and farm animals through establishment of oocyte banks [42]. However, damage of the cytoskeleton fibers (CSF) of oocytes during vitrification is the main cause of abnormal spindle configuration and reduced viability of frozen–thawed oocytes [29]. Recently, it was reported that the stabilization of CSF before vitrification could be beneficial for reducing CSF damage in oocytes [24]. In fact, several cytoskeleton stabilizing agents, such as cytochalasin B and D (CB, CD) [40,49] and paclitaxel (TaxolÒ) [38], have been used to protect oocytes from CSF damage during vitrification. Furthermore, it is well
⇑ Corresponding author. Fax: +66 4320 2362. E-mail address: [email protected] (T. Vongpralub).
documented that preincubation of matured oocytes with paclitaxel can reduce CSF damage after vitrification-warming and improve embryo development in humans [19], mice [31], pigs [38] and cattle [36]. Docetaxel is a recently identified member of a class of anti-cancer drugs, taxane diterpenoids, which also includes paclitaxel [5]. However, the modes of action of these two drugs are different. Docetaxel has been shown to promote both the rate and extent of tubulin polymerization into stable microtubules (MT) and inhibit MT depolymerization when exposed to ultra-low temperature; the rate and extent of tubulin polymerization promoted by docetaxel were twice those of paclitaxel [41]. Furthermore, the effective affinity of docetaxel for the MT binding site is 1.9-fold greater than that of paclitaxel [20]. In addition, docetaxel induces a twofold greater decrease of the critical concentration of GTP-tubulin required for tubulin polymerization when compared with paclitaxel [20,9].
http://dx.doi.org/10.1016/j.cryobiol.2015.07.002 0011-2240/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: J. Chasombat et al., Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.07.002
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Taken together, docetaxel may be more efficient than paclitaxel at stabilizing CSF of oocytes during vitrification and improving their viability after vitrification-warming. However, the toxicity of docetaxel on bovine oocytes has never been investigated. Furthermore, the effects of pretreatment of in vitro matured (IVM) bovine oocytes with docetaxel before vitrification on CSF integrity and oocyte survival after vitrification-warming, and rates of their subsequent embryo development, have not been reported. Therefore, the present study was conducted to investigate: (1) the effects of various doses of docetaxel in the preincubation medium on IVF of bovine oocytes; (2) the effects of preincubation with docetaxel before vitrification on CSF integrity of oocytes; and (3) the effects of preincubation with docetaxel before vitrification on oocyte survival after warming, and their rates of cleavage and subsequent development to the blastocyst stage after IVF. 2. Materials and methods Unless otherwise stated, all chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA). Tissue culture medium (TCM-199; HEPES buffer with Earle’s salts and sodium bicarbonate) and fetal calf serum (FCS) were obtained from Life Technologies (Carlsbad, CA, USA).Stock solutions of docetaxel (01885; Sigma–Aldrich) were prepared at 100 mM concentration in dimethylsulfoxide (DMSO). 2.1. Oocyte collection Bovine (Bos indicus) ovaries were obtained from a local abattoir and transported to the laboratory in normal saline solution (0.9% NaCl with 0.1 g/L streptomycin) at ambient temperature. The cumulus-oocyte complexes (COCs) were recovered by aspiration from antral follicles (2–6 mm) using an 18-gauge needle connected to a 10 mL syringe containing modified Dulbecco’s Phosphate Buffered Saline (mDPBS) with 10% FCS. The ovarian follicular fluid was pooled in 50 mL conical tubes and allowed to settle to the bottom of the tube during a 5 min interval. The COCs were selected under a stereomicroscope and washed five times with mDPBS with 10% FCS; those with more than two or three layers of cumulus cells and uniform cytoplasm (grades A and B) were subjected to in vitro maturation (IVM) [33]. 2.2. IVM of oocytes COCs were cultured using the procedure previously described [34]. Briefly, the COCs were washed three times with mDPBS supplemented with 10% FCS and three times with TCM-199 supplemented with 20% FCS. Groups of 20 COCs were then cultured for 24 h in 100 lL droplets of IVM medium (TCM-199 supplemented with 20% FCS, 10 lg/mL luteinizing hormone (LH), 1 lg/mL estradiol (E2), 0.5 lg/mL follicle stimulating hormone (FSH), 50 IU/mL penicillin G sodium and 50 mg/mL streptomycin) at 38.5 °C under a humidified atmosphere of 5% CO2 in air. 2.3. Vitrification and warming of IVM oocytes Vitrification and warming of IVM oocytes were performed according to the method previously described by Dinnyés et al. [14]. Briefly, cumulus cells of IVM oocytes were partially removed by gentle pipetting with a pulled pipette using 0.1% hyaluronidase in TCM-199. Subsequently, oocytes were washed three times with basic medium (BM) consisting of TCM-199 supplemented with 20% FCS; groups of five oocytes were then placed in an equilibration medium consisting of 4% ethylene glycol (EG) [29] in BM supplemented with 20% FCS at 39 °C for 12–15 min. Thereafter, oocytes
were rinsed three times in 20 lL droplets of vitrification solution consisting of 35% EG, 5% polyvinylpyrrolidone (PVP) and 0.4 M trehalose in BM for 25–30 s. They were then dropped as 1–2 lL droplets directly onto the surface of a steel cube that had been covered with aluminum foil and cooled to around 150 to 180 °C by partial immersion in liquid nitrogen (LN2). The droplets were instantaneously vitrified. Using nitrogen-cooled forceps, the vitrified droplets were moved into 1.5 mL LN2-filled cryovials and stored in a LN2 tank for 3 weeks. Thereafter, the vitrified droplets containing oocytes in cryovials were moved immediately by using nitrogen-cooled forceps into a 35 mm petri dish containing with BM supplemented with 0.3 M trehalose at 39 °C for 2 min, followed by treatment with BM supplemented with 0.15, 0.075 and 0.0375 M trehalose for 1 min each. Then, the retrieved oocyte were washed and transferred to BM until used in the following procedures. 2.4. Evaluation of oocyte survival after vitrification-warming At 2 h after warming, survival of vitrified oocytes was evaluated by fluorescein diacetate (FDA) staining, according to the method described by Mohr and Trounson [28]. Briefly, oocytes were treated with 2.5 lg/mL FDA in mDPBS supplemented with 5 mg/mL bovine serum albumin (BSA) at 38.5 °C for 2 min in darkness and then washed three times with mDPBS supplemented with 5 mg/mL BSA. Oocytes were evaluated under a fluorescence microscope (IX-71; Olympus, Tokyo, Japan) with UV irradiation using a U-MWIB3 filter with an excitation wavelength of 460–495 nm and emission at 510 nm. Oocytes expressing bright green fluorescence were regarded as living and were used in subsequent experiments. 2.5. Microtubule (MT) and chromosome (CM) staining Microtubule (MT) and chromosome (CM) staining were performed according to the method previously described by Adona et al. [3]. Briefly, at 2 h after warming, oocytes freed from cumulus cells were fixed in 3% paraformaldehyde supplemented with 0.6% Triton X-100 in mDPBS with 0.1% polyvinyl alcohol (PP) [37] for 30 min. Subsequently, they were washed three times in PP and blocked in 3% goat serum in PP for 45 min. Then, oocytes were stained with FITC-conjugated anti-a-tubulin antibody (1:100 in PP) for 1 h. After staining, oocytes were washed three times with PP and stained with 10 lg/mL propidium iodide (PI) for 15 min, then washed in PP and mounted between a slide and a coverslip in glycerol. MT and CM morphology was observed under fluorescence microscopy. MT morphology was classified into two categories – (1) normal: barrel-shaped, with chromosomes clustered as a discrete bundle at the metaphase plate and MT crossing the length of the spindle from pole to pole, or extending from the spindle poles to the chromosomes. MT within the polar body had no discernible organization and instead appeared as an amorphous mass intertwined with chromatin occupying the perivitelline space. (2) Abnormal: MT were not organized into typical spindles, or some were disassembled. Details of MT patterns found are provided in Fig. 1. CM organization was classified into two categories – (1) dispersed: CM were scattered throughout the cytoplasm or dispersed in a few zones of the cytoplasm; (2) condensed: CM with an aberrant, less condensed appearance. Details of CM patterns found are provided in Fig. 1. 2.6. Staining of cortical granules (CG) and mitochondria (MC) Cortical granules (CG) were detected following a protocol described previously [8], with modifications. Briefly,
Please cite this article in press as: J. Chasombat et al., Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.07.002
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Fig. 1. Microtubules (MT) and chromosomes (CM) in vitrified-warmed MII bovine oocytes preincubated with docetaxel before vitrification. Note the presence of the polar body (PB). The arrows indicate CM (in red; PI) and MT (in green; FITC-conjugated anti-a-tubulin antibody). (A) Normal MT and CM: barrel-shaped with CM clustered as a discrete bundle at the metaphase plate and MT crossing the length of the spindle from pole to pole, or extending from the spindle poles to the CM. (B) Abnormal MT and CM: MT are not organized as typical spindles, or some MT are disassembled and CM are condensed. Scale bar represents 20 lM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
cumulus-free oocytes were freed from the zona pellucida with 0.5% pronase in mDPBS and fixed in 3% paraformaldehyde in PP for 30 min. Thereafter, oocytes were blocked for 2 h in mDPBS with 0.1% BSA, 0.75% glycine and 0.2% sodium azide. Then the oocytes were permeabilized with 0.1% Triton X-100 in blocking solution (BS) for 5 min and washed three times in BS. The oocytes were then stained with 1 lg/ml FITC-conjugated Lensculinaris lectin and 10 lg/ml PI in BS for 15 min, washed three times with BS, and mounted between a glass slide and a coverslip in glycerol for evaluation under a fluorescence microscope. For detection of mitochondria (MC) distribution, oocytes were stained with 0.5 lM MitoTrackerÒ Red (Life Technologies, Carlsbad, CA, USA) and 10 lg/mL Hoechst 33342 in PP for 20 min, washed three times with PP, mounted and evaluated as described. The criteria used to define the CG morphology of oocytes at the metaphase of the second meiosis (MII) have been described elsewhere [48] – (1) normal: CG were distributed adjacent to the plasma membrane and positioned such that they formed a monolayer; (2) abnormal: most of the CG were distributed in the cortical area away from the plasma membrane and they did not form a
monolayer. Details of the different patterns found are provided in Fig. 2. Classification of MC distribution was performed by the method described by Katayama et al. [25]. Several patterns of MitoTracker signals were observed and classified into the following two groups – (1) normal distribution: MitoTracker signals were around lipid droplets that were identified by their dark (unstained) regular spot-like appearance; (2) abnormal distribution: various-sized cavities (no MitoTracker signals) were observed at the entire cortex of oocytes, no or weak signals at part of the oocyte cortex. Details of the different patterns found are provided in Fig. 3. 2.7. Evaluation of inner cell mass (ICM) and trophectoderm (TE) cell numbers of blastocysts Blastocysts appearing on day 7 (day 0 = day of IVF) were subjected to a differential staining protocol [44], with modifications. Briefly, blastocysts were washed three times with PP and then treated with 0.1 mg/mL PI and 0.2% (v/v) Triton-X 100 in mDPBS for 1 min to stain the TE cells. The ICM cells were then labeled with
Fig. 2. Cortical granules (CG) invitrified-warmed MII bovine oocytes preincubated with docetaxel before vitrification. Note the presence of the polar body (PB). The arrows indicate CG (in green; FITC-conjugated Lensculinaris lectin). (A) Normal CG: CG are distributed adjacent to the plasma membrane and are positioned such that they form a monolayer. (B) Abnormal CG: CG are aggregated. Scale bar represents 20 lM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: J. Chasombat et al., Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.07.002
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Fig. 3. Mitochondria (MC) distribution in vitrified-warmed MII bovine oocytes preincubated with docetaxel before vitrification. MC are stained red (MitoTracker Red). (A) Normal MC: MC are fully distributed in the cytoplasm and can be identified by their dark (unstained) regular spot-like appearance. (B) Abnormal MC: MC are aggregated. Scale bar represents 20 lM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
25 lg/mL Hoechst 33342 dissolved in 99.5% ethanol for 5 min. The stained embryos were subsequently washed in glycerol and mounted on a glass microscope slide, flattened in glycerol by a cover slip to a level where all nuclei appeared at the same focal plane, and visualized using a fluorescence microscope under UV light with excitation at 330–385 nm and emission at 420 nm. A digital image of each embryo was captured, and the numbers of TE (pink) and ICM (blue) nuclei were counted using NIH ImageJ (v.1.40) software [1].
stereo zoom microscopy. The embryos, developed to the two-cell stage or beyond, were washed twice in C-mSOFaa (mSOFaa with 0.5 mg/mL glucose, 1 lg/mL citrate and 3 mg/mL BSA), transferred immediately into 100 lL droplets of C-mSOFaa with bovine oviductal epithelial cells covered with mineral oil, and cultured in a humidified atmosphere of 5% CO2 in air for an additional 5 days (7 days in total). The culture medium was replaced with fresh medium every other day. The cleavage and blastocyst formation rates on day 2 and day 7, respectively, were recorded.
2.8. IVF
2.10. Experimental design
Sperm used for IVF were prepared using the swim-up method according to Parrish [32], with minor modifications. Briefly, frozen Thai native bull semen was thawed at 37 °C in a water bath for 30 s. The swim-up method was carried out by placing 200 lL of the semen into the bottom of a 5 mL tube containing 2 mL Tyrode’s albumin lactate pyruvate-HEPES (TALP-HEPES) medium supplemented with 0.3% fatty acid-free BSA and incubated at 38.5 °C in a humidified atmosphere of 5% CO2 for 1 h to allow live spermatozoa to swimup. After that, 1 mL of the upper phase of the medium was transferred to a 15 mL tube and centrifuged at 800g at 38 °C for 5 min. The supernatant was removed and sperm pellets were re-suspended in TALP-HEPES medium and washed twice at 485g for 5 min. Thereafter, the supernatant was removed again and sperm pellets were re-suspended in 1.5 mL TALP-IVF medium consisting of TALP supplemented with 0.3% fatty acid-free BSA, 3.0 lg/mL heparin and 3.0 lg/mL penicillamine. The solution was diluted to a final sperm concentration of 3 106 sperms/mL before IVF. In the IVF procedure, after 24 h of IVM, oocytes were washed three times with TALP-IVF medium; then a group of 20 COCs was placed in 100 lL/droplets of TALP-IVF medium and incubated with sperms at 38.5 °C in a humidified atmosphere of 5% CO2 in air for 18 h [50].
2.10.1. Experiment 1: Effect of preincubation of IVM bovine oocytes with various doses of docetaxel on IVF and subsequent embryo development In this experiment, IVM oocytes were randomly selected into five treatment groups – group 1: IVM oocytes were untreated and subjected to IVF (control); groups 2–5: IVM oocytes were preincubated for 30 min with 0.05, 0.5, 5.0 and 50.0 lM of docetaxel, respectively. Thereafter, the oocytes from each treatment group were subjected to IVF.
2.9. In vitro culture (IVC) The embryos were cultured in modified synthetic oviductal fluid with amino acid (mSOFaa) [22]. Briefly, approximately 18– 20 h after insemination, the embryos were washed four times in mSOFaa and once in G-mSOFaa (mSOFaa with 0.5 lg/mL glutamine and 3 mg/mL BSA). Subsequently, the embryos were transferred into 100 lL droplets of G-mSOFaa covered with mineral oil and cultured for 2 days at 38.5 °C in an atmosphere of 5% CO2, 5% O2 and 90% N2. The cleavage rates were determined on day 2 under
2.10.2. Experiment 2: Effect of preincubation of IVM bovine oocytes with docetaxel before vitrification on their cytoskeletal integrity after vitrification-warming Based on the results of experiment 1, docetaxel at a concentration of 0.05 lM was used in this experiment. There were five treatment groups – group 1: IVM oocytes were untreated (Fresh control); group 2: IVM oocytes were preincubated with docetaxel for 30 min (Docetaxel group); group 3: IVM oocytes were preincubated with docetaxel for 30 min and exposed to cryoprotectants (CPAs) in vitrification solution but not vitrified (Docetaxel-CPAs group); group 4: IVM oocytes were vitrified by the solid surface vitrification (SSV) method (SSV group); and group 5: IVM oocytes were preincubated with docetaxel for 30 min and subjected to vitrification by the SSV method (Docetaxel-SSV group). All oocytes (vitrified oocytes after warming) were evaluated for the morphology of MT, CM, CG and MC by the methods described above. 2.10.3. Experiment 3: Effect of preincubation of IVM bovine oocytes with docetaxel before vitrification on their survival after vitrificationwarming and subsequent embryo development after IVF In this experiment, the treatment regimen was the same as in experiment 2 except that all the oocytes were subjected to IVF. The presumed zygotes were placed in culture and allowed to
Please cite this article in press as: J. Chasombat et al., Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.07.002
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vitrification (Docetaxel + SSV group) compared with those not preincubated with docetaxel before vitrification (SSV group) (P < 0.05). In fact, the proportion of oocytes with normal MT configuration was significantly higher and that of oocytes with dispersed CM alignment was significantly lower in the fresh control and in the toxicity solution control groups (Docetaxel and Docetaxel + CPAs groups) than in the vitrification groups (SSV and Docetaxel-SSV groups) (P < 0.05). Details of MT configuration and CM alignment patterns are provided in Fig. 1. Besides, the proportion of oocytes with normal CG and MC distribution was significantly higher in the Docetaxel + SSV group than in the SSV group (P < 0.05). Moreover, there was no significant difference in the proportion of oocytes with normal CG and MC distribution among the Docetaxel + SSV, fresh control, and toxicity solution control groups (Docetaxel and Docetaxel + CPAs groups), as shown in Table 4. The details of CG and MC and distribution patterns are provided in Figs. 2 and 3.
develop for 7 days, and their cleavage and blastocyst formation rates were recorded. 2.11. Statistics Data were analyzed statistically using the Statistical Analysis System version 9 (SAS Institute, Cary, NC, USA). A v2 test was used to compare data among the experimental groups. P-values of
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming Jakkhaphan Chasombat a, Takashi Nagai b,c, Rangsun Parnpai d, Thevin Vongpralub a,⇑ a
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand Food and Fertilizer Technology Center, Taipei 10648, Taiwan NARO Institute of Livestock and Grassland Science, Tsukuba, Japan d Embryo Technology and Stem Cell Research Center and School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand b c
a r t i c l e
i n f o
Article history: Received 28 March 2015 Revised 16 June 2015 Accepted 9 July 2015 Available online xxxx Keywords: Docetaxel Bovine oocytes Vitrification Cytoskeleton fibers
a b s t r a c t The stabilization of spindle fibersis important for successful vitrification of bovine oocytes because microtubules and other cytoskeleton fibers (CSF) can be damaged during vitrification, resulting in failure of fertilization after thawing. Docetaxel, a stabilizing agent, could potentially reduce CSF damage of bovine oocytes induced during vitrification. However, there have been no reports on the effects of docetaxel on their vitrification. Experiment 1 was conducted to investigate the effects of various doses of docetaxel (0.0, 0.05, 0.5, 5.0 and 50 lM) in preincubation medium of in vitro matured (IVM) bovine oocytes on their developmental ability after in vitro fertilization (IVF). The results show that 0.05 lM docetaxel had no adverse effect on embryo development, while docetaxel at a concentration of P0.5 lM inhibited development. Experiments 2 and 3 were conducted to investigate the effects of preincubation of IVM bovine oocytes with 0.05 lM docetaxel for 30 min prior to vitrification-warming on CSF integrity (Experiment 2), and on oocyte survival and viability after IVF (Experiment 3). When preincubated with 0.05 lM docetaxel for 30 min before vitrification, post-thawed oocytes had less CSF damage and higher survival rates compared with those untreated with docetaxel before vitrification. Surviving oocytes also had higher rates of cleavage and development to the blastocyst stage after IVF. In conclusion, preincubation of IVM bovine oocytes with 0.05 lM docetaxel for 30 min before vitrification was effective at preventing CSF damage during vitrification, and improving oocyte viability after warming and subsequent cleavage and blastocyst formation after IVF. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Vitrification of oocytes is a powerful method for preservation of genetic diversity in endangered animal species and farm animals through establishment of oocyte banks [42]. However, damage of the cytoskeleton fibers (CSF) of oocytes during vitrification is the main cause of abnormal spindle configuration and reduced viability of frozen–thawed oocytes [29]. Recently, it was reported that the stabilization of CSF before vitrification could be beneficial for reducing CSF damage in oocytes [24]. In fact, several cytoskeleton stabilizing agents, such as cytochalasin B and D (CB, CD) [40,49] and paclitaxel (TaxolÒ) [38], have been used to protect oocytes from CSF damage during vitrification. Furthermore, it is well
⇑ Corresponding author. Fax: +66 4320 2362. E-mail address: [email protected] (T. Vongpralub).
documented that preincubation of matured oocytes with paclitaxel can reduce CSF damage after vitrification-warming and improve embryo development in humans [19], mice [31], pigs [38] and cattle [36]. Docetaxel is a recently identified member of a class of anti-cancer drugs, taxane diterpenoids, which also includes paclitaxel [5]. However, the modes of action of these two drugs are different. Docetaxel has been shown to promote both the rate and extent of tubulin polymerization into stable microtubules (MT) and inhibit MT depolymerization when exposed to ultra-low temperature; the rate and extent of tubulin polymerization promoted by docetaxel were twice those of paclitaxel [41]. Furthermore, the effective affinity of docetaxel for the MT binding site is 1.9-fold greater than that of paclitaxel [20]. In addition, docetaxel induces a twofold greater decrease of the critical concentration of GTP-tubulin required for tubulin polymerization when compared with paclitaxel [20,9].
http://dx.doi.org/10.1016/j.cryobiol.2015.07.002 0011-2240/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: J. Chasombat et al., Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.07.002
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Taken together, docetaxel may be more efficient than paclitaxel at stabilizing CSF of oocytes during vitrification and improving their viability after vitrification-warming. However, the toxicity of docetaxel on bovine oocytes has never been investigated. Furthermore, the effects of pretreatment of in vitro matured (IVM) bovine oocytes with docetaxel before vitrification on CSF integrity and oocyte survival after vitrification-warming, and rates of their subsequent embryo development, have not been reported. Therefore, the present study was conducted to investigate: (1) the effects of various doses of docetaxel in the preincubation medium on IVF of bovine oocytes; (2) the effects of preincubation with docetaxel before vitrification on CSF integrity of oocytes; and (3) the effects of preincubation with docetaxel before vitrification on oocyte survival after warming, and their rates of cleavage and subsequent development to the blastocyst stage after IVF. 2. Materials and methods Unless otherwise stated, all chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA). Tissue culture medium (TCM-199; HEPES buffer with Earle’s salts and sodium bicarbonate) and fetal calf serum (FCS) were obtained from Life Technologies (Carlsbad, CA, USA).Stock solutions of docetaxel (01885; Sigma–Aldrich) were prepared at 100 mM concentration in dimethylsulfoxide (DMSO). 2.1. Oocyte collection Bovine (Bos indicus) ovaries were obtained from a local abattoir and transported to the laboratory in normal saline solution (0.9% NaCl with 0.1 g/L streptomycin) at ambient temperature. The cumulus-oocyte complexes (COCs) were recovered by aspiration from antral follicles (2–6 mm) using an 18-gauge needle connected to a 10 mL syringe containing modified Dulbecco’s Phosphate Buffered Saline (mDPBS) with 10% FCS. The ovarian follicular fluid was pooled in 50 mL conical tubes and allowed to settle to the bottom of the tube during a 5 min interval. The COCs were selected under a stereomicroscope and washed five times with mDPBS with 10% FCS; those with more than two or three layers of cumulus cells and uniform cytoplasm (grades A and B) were subjected to in vitro maturation (IVM) [33]. 2.2. IVM of oocytes COCs were cultured using the procedure previously described [34]. Briefly, the COCs were washed three times with mDPBS supplemented with 10% FCS and three times with TCM-199 supplemented with 20% FCS. Groups of 20 COCs were then cultured for 24 h in 100 lL droplets of IVM medium (TCM-199 supplemented with 20% FCS, 10 lg/mL luteinizing hormone (LH), 1 lg/mL estradiol (E2), 0.5 lg/mL follicle stimulating hormone (FSH), 50 IU/mL penicillin G sodium and 50 mg/mL streptomycin) at 38.5 °C under a humidified atmosphere of 5% CO2 in air. 2.3. Vitrification and warming of IVM oocytes Vitrification and warming of IVM oocytes were performed according to the method previously described by Dinnyés et al. [14]. Briefly, cumulus cells of IVM oocytes were partially removed by gentle pipetting with a pulled pipette using 0.1% hyaluronidase in TCM-199. Subsequently, oocytes were washed three times with basic medium (BM) consisting of TCM-199 supplemented with 20% FCS; groups of five oocytes were then placed in an equilibration medium consisting of 4% ethylene glycol (EG) [29] in BM supplemented with 20% FCS at 39 °C for 12–15 min. Thereafter, oocytes
were rinsed three times in 20 lL droplets of vitrification solution consisting of 35% EG, 5% polyvinylpyrrolidone (PVP) and 0.4 M trehalose in BM for 25–30 s. They were then dropped as 1–2 lL droplets directly onto the surface of a steel cube that had been covered with aluminum foil and cooled to around 150 to 180 °C by partial immersion in liquid nitrogen (LN2). The droplets were instantaneously vitrified. Using nitrogen-cooled forceps, the vitrified droplets were moved into 1.5 mL LN2-filled cryovials and stored in a LN2 tank for 3 weeks. Thereafter, the vitrified droplets containing oocytes in cryovials were moved immediately by using nitrogen-cooled forceps into a 35 mm petri dish containing with BM supplemented with 0.3 M trehalose at 39 °C for 2 min, followed by treatment with BM supplemented with 0.15, 0.075 and 0.0375 M trehalose for 1 min each. Then, the retrieved oocyte were washed and transferred to BM until used in the following procedures. 2.4. Evaluation of oocyte survival after vitrification-warming At 2 h after warming, survival of vitrified oocytes was evaluated by fluorescein diacetate (FDA) staining, according to the method described by Mohr and Trounson [28]. Briefly, oocytes were treated with 2.5 lg/mL FDA in mDPBS supplemented with 5 mg/mL bovine serum albumin (BSA) at 38.5 °C for 2 min in darkness and then washed three times with mDPBS supplemented with 5 mg/mL BSA. Oocytes were evaluated under a fluorescence microscope (IX-71; Olympus, Tokyo, Japan) with UV irradiation using a U-MWIB3 filter with an excitation wavelength of 460–495 nm and emission at 510 nm. Oocytes expressing bright green fluorescence were regarded as living and were used in subsequent experiments. 2.5. Microtubule (MT) and chromosome (CM) staining Microtubule (MT) and chromosome (CM) staining were performed according to the method previously described by Adona et al. [3]. Briefly, at 2 h after warming, oocytes freed from cumulus cells were fixed in 3% paraformaldehyde supplemented with 0.6% Triton X-100 in mDPBS with 0.1% polyvinyl alcohol (PP) [37] for 30 min. Subsequently, they were washed three times in PP and blocked in 3% goat serum in PP for 45 min. Then, oocytes were stained with FITC-conjugated anti-a-tubulin antibody (1:100 in PP) for 1 h. After staining, oocytes were washed three times with PP and stained with 10 lg/mL propidium iodide (PI) for 15 min, then washed in PP and mounted between a slide and a coverslip in glycerol. MT and CM morphology was observed under fluorescence microscopy. MT morphology was classified into two categories – (1) normal: barrel-shaped, with chromosomes clustered as a discrete bundle at the metaphase plate and MT crossing the length of the spindle from pole to pole, or extending from the spindle poles to the chromosomes. MT within the polar body had no discernible organization and instead appeared as an amorphous mass intertwined with chromatin occupying the perivitelline space. (2) Abnormal: MT were not organized into typical spindles, or some were disassembled. Details of MT patterns found are provided in Fig. 1. CM organization was classified into two categories – (1) dispersed: CM were scattered throughout the cytoplasm or dispersed in a few zones of the cytoplasm; (2) condensed: CM with an aberrant, less condensed appearance. Details of CM patterns found are provided in Fig. 1. 2.6. Staining of cortical granules (CG) and mitochondria (MC) Cortical granules (CG) were detected following a protocol described previously [8], with modifications. Briefly,
Please cite this article in press as: J. Chasombat et al., Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.07.002
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Fig. 1. Microtubules (MT) and chromosomes (CM) in vitrified-warmed MII bovine oocytes preincubated with docetaxel before vitrification. Note the presence of the polar body (PB). The arrows indicate CM (in red; PI) and MT (in green; FITC-conjugated anti-a-tubulin antibody). (A) Normal MT and CM: barrel-shaped with CM clustered as a discrete bundle at the metaphase plate and MT crossing the length of the spindle from pole to pole, or extending from the spindle poles to the CM. (B) Abnormal MT and CM: MT are not organized as typical spindles, or some MT are disassembled and CM are condensed. Scale bar represents 20 lM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
cumulus-free oocytes were freed from the zona pellucida with 0.5% pronase in mDPBS and fixed in 3% paraformaldehyde in PP for 30 min. Thereafter, oocytes were blocked for 2 h in mDPBS with 0.1% BSA, 0.75% glycine and 0.2% sodium azide. Then the oocytes were permeabilized with 0.1% Triton X-100 in blocking solution (BS) for 5 min and washed three times in BS. The oocytes were then stained with 1 lg/ml FITC-conjugated Lensculinaris lectin and 10 lg/ml PI in BS for 15 min, washed three times with BS, and mounted between a glass slide and a coverslip in glycerol for evaluation under a fluorescence microscope. For detection of mitochondria (MC) distribution, oocytes were stained with 0.5 lM MitoTrackerÒ Red (Life Technologies, Carlsbad, CA, USA) and 10 lg/mL Hoechst 33342 in PP for 20 min, washed three times with PP, mounted and evaluated as described. The criteria used to define the CG morphology of oocytes at the metaphase of the second meiosis (MII) have been described elsewhere [48] – (1) normal: CG were distributed adjacent to the plasma membrane and positioned such that they formed a monolayer; (2) abnormal: most of the CG were distributed in the cortical area away from the plasma membrane and they did not form a
monolayer. Details of the different patterns found are provided in Fig. 2. Classification of MC distribution was performed by the method described by Katayama et al. [25]. Several patterns of MitoTracker signals were observed and classified into the following two groups – (1) normal distribution: MitoTracker signals were around lipid droplets that were identified by their dark (unstained) regular spot-like appearance; (2) abnormal distribution: various-sized cavities (no MitoTracker signals) were observed at the entire cortex of oocytes, no or weak signals at part of the oocyte cortex. Details of the different patterns found are provided in Fig. 3. 2.7. Evaluation of inner cell mass (ICM) and trophectoderm (TE) cell numbers of blastocysts Blastocysts appearing on day 7 (day 0 = day of IVF) were subjected to a differential staining protocol [44], with modifications. Briefly, blastocysts were washed three times with PP and then treated with 0.1 mg/mL PI and 0.2% (v/v) Triton-X 100 in mDPBS for 1 min to stain the TE cells. The ICM cells were then labeled with
Fig. 2. Cortical granules (CG) invitrified-warmed MII bovine oocytes preincubated with docetaxel before vitrification. Note the presence of the polar body (PB). The arrows indicate CG (in green; FITC-conjugated Lensculinaris lectin). (A) Normal CG: CG are distributed adjacent to the plasma membrane and are positioned such that they form a monolayer. (B) Abnormal CG: CG are aggregated. Scale bar represents 20 lM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: J. Chasombat et al., Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.07.002
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Fig. 3. Mitochondria (MC) distribution in vitrified-warmed MII bovine oocytes preincubated with docetaxel before vitrification. MC are stained red (MitoTracker Red). (A) Normal MC: MC are fully distributed in the cytoplasm and can be identified by their dark (unstained) regular spot-like appearance. (B) Abnormal MC: MC are aggregated. Scale bar represents 20 lM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
25 lg/mL Hoechst 33342 dissolved in 99.5% ethanol for 5 min. The stained embryos were subsequently washed in glycerol and mounted on a glass microscope slide, flattened in glycerol by a cover slip to a level where all nuclei appeared at the same focal plane, and visualized using a fluorescence microscope under UV light with excitation at 330–385 nm and emission at 420 nm. A digital image of each embryo was captured, and the numbers of TE (pink) and ICM (blue) nuclei were counted using NIH ImageJ (v.1.40) software [1].
stereo zoom microscopy. The embryos, developed to the two-cell stage or beyond, were washed twice in C-mSOFaa (mSOFaa with 0.5 mg/mL glucose, 1 lg/mL citrate and 3 mg/mL BSA), transferred immediately into 100 lL droplets of C-mSOFaa with bovine oviductal epithelial cells covered with mineral oil, and cultured in a humidified atmosphere of 5% CO2 in air for an additional 5 days (7 days in total). The culture medium was replaced with fresh medium every other day. The cleavage and blastocyst formation rates on day 2 and day 7, respectively, were recorded.
2.8. IVF
2.10. Experimental design
Sperm used for IVF were prepared using the swim-up method according to Parrish [32], with minor modifications. Briefly, frozen Thai native bull semen was thawed at 37 °C in a water bath for 30 s. The swim-up method was carried out by placing 200 lL of the semen into the bottom of a 5 mL tube containing 2 mL Tyrode’s albumin lactate pyruvate-HEPES (TALP-HEPES) medium supplemented with 0.3% fatty acid-free BSA and incubated at 38.5 °C in a humidified atmosphere of 5% CO2 for 1 h to allow live spermatozoa to swimup. After that, 1 mL of the upper phase of the medium was transferred to a 15 mL tube and centrifuged at 800g at 38 °C for 5 min. The supernatant was removed and sperm pellets were re-suspended in TALP-HEPES medium and washed twice at 485g for 5 min. Thereafter, the supernatant was removed again and sperm pellets were re-suspended in 1.5 mL TALP-IVF medium consisting of TALP supplemented with 0.3% fatty acid-free BSA, 3.0 lg/mL heparin and 3.0 lg/mL penicillamine. The solution was diluted to a final sperm concentration of 3 106 sperms/mL before IVF. In the IVF procedure, after 24 h of IVM, oocytes were washed three times with TALP-IVF medium; then a group of 20 COCs was placed in 100 lL/droplets of TALP-IVF medium and incubated with sperms at 38.5 °C in a humidified atmosphere of 5% CO2 in air for 18 h [50].
2.10.1. Experiment 1: Effect of preincubation of IVM bovine oocytes with various doses of docetaxel on IVF and subsequent embryo development In this experiment, IVM oocytes were randomly selected into five treatment groups – group 1: IVM oocytes were untreated and subjected to IVF (control); groups 2–5: IVM oocytes were preincubated for 30 min with 0.05, 0.5, 5.0 and 50.0 lM of docetaxel, respectively. Thereafter, the oocytes from each treatment group were subjected to IVF.
2.9. In vitro culture (IVC) The embryos were cultured in modified synthetic oviductal fluid with amino acid (mSOFaa) [22]. Briefly, approximately 18– 20 h after insemination, the embryos were washed four times in mSOFaa and once in G-mSOFaa (mSOFaa with 0.5 lg/mL glutamine and 3 mg/mL BSA). Subsequently, the embryos were transferred into 100 lL droplets of G-mSOFaa covered with mineral oil and cultured for 2 days at 38.5 °C in an atmosphere of 5% CO2, 5% O2 and 90% N2. The cleavage rates were determined on day 2 under
2.10.2. Experiment 2: Effect of preincubation of IVM bovine oocytes with docetaxel before vitrification on their cytoskeletal integrity after vitrification-warming Based on the results of experiment 1, docetaxel at a concentration of 0.05 lM was used in this experiment. There were five treatment groups – group 1: IVM oocytes were untreated (Fresh control); group 2: IVM oocytes were preincubated with docetaxel for 30 min (Docetaxel group); group 3: IVM oocytes were preincubated with docetaxel for 30 min and exposed to cryoprotectants (CPAs) in vitrification solution but not vitrified (Docetaxel-CPAs group); group 4: IVM oocytes were vitrified by the solid surface vitrification (SSV) method (SSV group); and group 5: IVM oocytes were preincubated with docetaxel for 30 min and subjected to vitrification by the SSV method (Docetaxel-SSV group). All oocytes (vitrified oocytes after warming) were evaluated for the morphology of MT, CM, CG and MC by the methods described above. 2.10.3. Experiment 3: Effect of preincubation of IVM bovine oocytes with docetaxel before vitrification on their survival after vitrificationwarming and subsequent embryo development after IVF In this experiment, the treatment regimen was the same as in experiment 2 except that all the oocytes were subjected to IVF. The presumed zygotes were placed in culture and allowed to
Please cite this article in press as: J. Chasombat et al., Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.07.002
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vitrification (Docetaxel + SSV group) compared with those not preincubated with docetaxel before vitrification (SSV group) (P < 0.05). In fact, the proportion of oocytes with normal MT configuration was significantly higher and that of oocytes with dispersed CM alignment was significantly lower in the fresh control and in the toxicity solution control groups (Docetaxel and Docetaxel + CPAs groups) than in the vitrification groups (SSV and Docetaxel-SSV groups) (P < 0.05). Details of MT configuration and CM alignment patterns are provided in Fig. 1. Besides, the proportion of oocytes with normal CG and MC distribution was significantly higher in the Docetaxel + SSV group than in the SSV group (P < 0.05). Moreover, there was no significant difference in the proportion of oocytes with normal CG and MC distribution among the Docetaxel + SSV, fresh control, and toxicity solution control groups (Docetaxel and Docetaxel + CPAs groups), as shown in Table 4. The details of CG and MC and distribution patterns are provided in Figs. 2 and 3.
develop for 7 days, and their cleavage and blastocyst formation rates were recorded. 2.11. Statistics Data were analyzed statistically using the Statistical Analysis System version 9 (SAS Institute, Cary, NC, USA). A v2 test was used to compare data among the experimental groups. P-values of
