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Embryo |
Department of Animal Sciences4
Interdisciplinary Center for Biotechnology Research,5 Electron Microscopy Core Laboratory, University of Florida, Gainesville, Florida 32611
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, early development, heat shock, in vitro fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well documented that heat shock (albeit of a higher temperature than that disrupting function of embryos) induces alterations to the cytoskeleton. The specific cytoskeletal element most sensitive to heat shock varies with cell type. For example, exposure of mouse epithelial cells to elevated temperatures changed the organization of keratin filaments and actin filaments but had no effect on microtubules [15]. Similar results were observed in 9L cells, where heat shock caused collapse of microfilaments and intermediate filaments, but had only slight effects on microtubules [16]. In contrast, microtubules were disrupted by heat shock in Chinese hamster ovary cells [17] and mouse 3T3 cells [18].
The objective of the present study was to determine the involvement of disruption in the microtubule and microfilament cytoskeleton in heat-shock-induced redistribution of organelles and other ultrastructural changes in the bovine two-cell embryo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals, except as otherwise specified, were from Sigma (St. Louis, MO). Reagents for in vitro production of embryos were obtained as described elsewhere [4, 5]. Percoll was from Amersham Biosciences (Piscataway, NJ). Penicillin-streptomycin and SP-TL, HEPES-TL, and IVF-TL culture media were from Cell and Molecular Technologies (Lavallette, NJ) and were used to prepare SP-TALP, Hepes-TALP, and IVF- TALP [19]. Potassium simplex optimized medium (KSOM; formulation MR-020-D) was also from Cell and Molecular Technologies and was used to prepare KSOM-BE1 by the addition of 3 mg/ml essentially fatty acid- free Fraction V BSA, 2.5 µg/ml gentamicin, essential amino acids (basal medium Eagle), and nonessential amino acids (minimum essential medium) as described elsewhere [20]. Bovine steer serum was from Pel Freez Biologicals (Rogers, AK). Glutaraldehyde, 16% (v/v) formaldehyde, osmium tetroxide, ethanol, acetone, EMbed-812, uranyl acetate, Reynolds lead citrate, Butvar, and copper grids were from Electron Microscopy Sciences (Fort Washington, PA). Alexa Fluor 594 phalloidin and ProLong antifade solution were from Molecular Probes (Eugene, OR).
Production of Embryos
Procedures for oocyte maturation, fertilization, and embryo culture were as described previously [4, 5]. Briefly, oocytes obtained by slashing ovaries collected at an abattoir were matured for 21 h and then inseminated with a cocktail of Percoll-purified spermatozoa from three different Angus bulls; a different group of bulls was used for each replicate. At 18–20 h after insemination, putative zygotes were denuded of cumulus cells by suspension in Hepes-TALP medium containing 1000 U/ml of hyaluronidase type IV and vortexing in a microcentrifuge tube. Presumptive zygotes were then placed in groups of 
30 in 50-µl microdrops of KSOM-BE1. Two-cell embryos were selected at 28 h after insemination and placed in fresh 50-µl microdrops of KSOM-BE1 for experiments. All cultures at 38.5°C were performed in an environment of 5% (v/v) CO2 in humidified air. The percent CO2 was adjusted in the incubators used for heat shock treatments (7% and 8% CO2 for 41.0 and 43.0°C, respectively) to maintain medium pH at 7.4. The CO2 contents used for each temperature were determined experimentally to be those that maintained pH at 7.4. The temperature of all incubators was calibrated routinely to assure accuracy of treatments.
Effects of Microfilament and Microtubule Depolymerization on Heat-Shock-Induced Movement of Organelles
This experiment was designed as a 3 x 3 factorial design with three inhibitor treatments-vehicle (0.1% ethanol), 1 µM latrunculin B (a potent inhibitor of actin polymerization, which causes a shortening and thickening of stress fibers; [21]), and 10 nM rhizoxin (inhibits microtubule assembly and also depolymerizes preformed microtubules; [22]), and three temperatures: –38.5°C (i.e., homoeothermic body temperature of the cow), 41.0°C (characteristic temperature for heat-stressed cows), or 43.0°C (severe heat shock). Approximately 28 h postinsemination, two-cell embryos were collected and placed in fresh 50-µl microdrops of KSOM-BE1 with or without cytoskeletal inhibitors (5–10 embryos per drop). Embryos were cultured for 6 h at one of the three experimental temperatures. Immediately after treatment, embryos were processed for electron microscopy. The experiment was replicated three times on different days. For statistical analysis, electron micrographs of one embryo per replicate per treatment (selected at random) were used to take measurements on cellular morphology.
Effects of Microtubule Stabilization on Heat-Shock- Induced Movement of Organelles
This experiment used a 2 x 3 factorial design with inhibitor treatments consisting of vehicle (0.1% dimethyl sulphoxide; DMSO) or 1 µM paclitaxel (promotes the formation of highly stable microtubules that resist depolymerization; [23]) and temperatures of 38.5, 41.0, or 43.0°C. Two- cell embryos (5–10/replicate) were cultured in microdrops of KSOM-BE1 with or without paclitaxel for 6 h at one of the three temperatures listed above. Immediately after treatment, embryos were processed for electron microscopy. The experiment was replicated three times on different days. For statistical analysis, electron micrographs of one randomly selected embryo per replicate per treatment were used to take measurements on cellular morphology.
Electron Microscopy and Morphometric Analysis of Electron Micrographs
Procedures for embryo fixation and processing were conducted as described previously [5]. For each two-cell embryo, morphometric analysis was performed on four electron micrographs obtained from one randomly chosen blastomere. Morphometric analysis was performed using an SIS Megaview III camera and software (Soft Imaging System, Lakewood, CO). The distance from the plasma membrane to the organelle-containing region of the cytoplasm was measured at 10 random locations from each of four pictures per embryo (i.e., 40 random measurements/embryo). The average of these measurements was used for statistical analysis. Electron micrographs were also visually assessed to determine effects of heat shock on other aspects of cellular morphology.
Localization of Microfilament by Fluorescence Microscopy
Two-cell embryos were cultured at 38.5 or 43.0°C for 6 h. Immediately after heat shock, the zona pellucida of each embryo was removed by incubation in 0.5% (w/v) protease from Streptomyces griseus in phosphate-buffered saline (PBS; 10 mM; NaPO4, pH 7.4, containing 0.15 M NaCl) supplemented with 1 mg/ml polyvinyl pyrrolidone (PBS+PVP) for approximately 1 min at 39.0°C. Embryos were thoroughly washed in KSOM-BE1 before a 10-min fixation with 3.7% (v/v) formaldehyde in 0.2 M NaPO4, pH 7.4, containing 0.15 M NaCl at 39.0°C. After fixation, embryos were washed three times in PBS+PVP. Embryos were then permeabilized with 0.1% Triton-X 100 for 5 min followed by three washes with PBS+PVP. Embryos were incubated in 10% (v/v) normal goat serum in PBS (PBS+S) at room temperature for 30 min followed by incubation in Alexa Fluor 594 phalloidin for 30 min at room temperature. The phalloidin was prepared by diluting 5 µl methanolic phalloidin stock solution (prepared as per manufacturer's specification) in 200 L PBS+S. Embryos were then washed three times in PBS+S before a second 10-min fixation in 3.7% (v/v) formaldehyde, washing three times in PBS+PVP and placement on a glass slide with a cover slip mounted with 5 µl ProLong antifade solution. Negative controls were stained similarly except that phalloidin was omitted from the solution. Embryos were viewed using a high-resolution black and white AxioCam MRm digital camera connected to a Zeiss Axioplan2 epifluorescent microscope (Carl Zeiss, Gottingen, Germany). Images were acquired using AxioVision software (Zeiss). The experiment was replicated three times with a total of 15–16 embryos per treatment. Attempts were made to immunolocalize microtubules by the use of fluorescent microscopy with antitubulin but tubules could not be visualized unless mitotic spindles were present in the cell.
Statistical Analysis
Treatment effects on the distance from the plasma membrane to the outermost organelles were analyzed by least-squares analysis of variance using the Proc GLM procedures of SAS (SAS for Windows, Version 8e; Cary, NC). The model included effects of temperature, inhibitor treatment, and temperature x treatment. Data are presented as least-squares means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two-cell bovine embryos are mainly composed of immature mitochondria and vesicles of unknown composition (Fig. 1). For embryos cultured at 38.5°C, organelles were evenly distributed throughout the blastomere and extended to the plasma membrane, regardless of treatment (Fig. 1, A–C). Heat shock at 41.0 and 43.0°C resulted in movement of organelles away from the periphery of the blastomere to create an area devoid of organelles (Fig. 1, D and G). Other effects of heat shock were also evident. For example, a proportion of the mitochondria were swollen at both heat- shock temperatures (e.g., see Fig. 2B) and the cytoplasm of embryos cultured at 43.0°C had electron dense material (Fig. 2D). This material was throughout the cytoplasm (Figs. 1, G–I, and 2D) and especially in close proximity to the membranes of organelles, except vesicles. Precipitated chromatin could also be observed for embryos at 43.0°C (Fig. 2F).
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Latrunculin B was effective in causing microfilament disruption as determined by fluorescence microscopy of phalloidin-labeled microfilaments (Fig. 3). The morphology of microfilaments was changed from long, irregularly shaped thin fibers (Fig. 3A) to aggregated material with numerous nodule-like structures (Fig. 3, B and C). A large ring of actin-containing fibers was also present in some blastomeres from latrunculin B-treated embryos (Fig. 3B). While embryos cultured in vehicle possessed an intense, narrow network of microfilaments underneath the plasma membrane (Fig. 3A), this network was no longer evident in embryos cultured with latrunculin B (Fig. 3, B and C).
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The microfilament and microtubule depolymerization agents affected the movement of organelles as a result of heat shock. Results of quantitative analysis of the distance between the plasma membrane and the nearest organelle are illustrated in Figure 4, while representative electron micrographs illustrating the phenomenon are shown in Figure 1, D–I. Both depolymerization agents reduced the heat- shock-induced movement of organelles (temperature x treatment interaction, P < 0.001). However, latrunculin B blocked the effect of heat shock at 41.0°C but not at 43.0°C (see Fig. 1, B, E, and H), while rhizoxin blocked the movement of organelles at both temperatures (Fig. 1, C, F, and I).
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Rhizoxin, but not latrunculin B, also altered mitochondrial changes induced by heat shock. In particular, the increase in the number of swollen mitochondria caused by heat shock at both 41.0 and 43.0°C was absent for embryos treated with rhizoxin.
Effects of Microtubule Stabilization on Heat-Induced Movement of Organelles
Representative electron micrographs of embryos treated with paclitaxel or vehicle (0.1% DMSO) are shown in Figure 5. As for the previous experiment, heat shock caused organelles to redistribute away from the plasma membrane (Fig. 5, A, C, and E) and increased the proportion of mitochondria with a swollen morphology. Furthermore, several of the embryos at 43.0°C (but not at 38.5 or 41.0°C) possessed structures resembling intranuclear intermediate filament bundles (Fig. 6).
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Treatment with paclitaxel caused changes in the ultrastructure of embryos at 38.5°C, consistent with its action as a stabilizer of microtubules. In the absence of paclitaxel, the occasional microtubule was observed in the cytoplasm (Fig. 7A). In contrast, embryos treated with paclitaxel possessed numerous macrotubules (bundles of microtubules) in the cytoplasm (Fig. 7C). Tubulin paracrystals were also apparent (Fig. 7E). In addition, paclitaxel caused alignment of mitochondria into vertical columns organized along the nuclear-plasma membrane axis (Figs. 5B and 8, E–F). This observation was unlike other treatments at 38.5°C (vehicle, latrunculin B, or rhizoxin), where mitochondria were evenly distributed throughout the cytoplasm (Fig. 8, A–D). Further, paclitaxel caused accumulation of organelles at the cell margin (Fig. 5B) and blastomere fragmentation (Fig. 7, D and F).
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Results of quantitative analysis to determine whether paclitaxel affected the actions of heat shock to cause organellar movement is shown in Figure 9, while representative electron micrographs illustrating the phenomenon are shown in Figure 5. The movement of organelles was progressively increased by heat shock in a temperature-dependent manner (temperature; P < 0.01). While addition of paclitaxel reduced the distance between the plasma membrane and the nearest organelle (P < 0.01), the inhibitor did not prevent movement of organelles away from the plasma membrane in response to heat shock (there was no temperature x treatment interaction). Paclitaxel also did not prevent the induction of mitochondrial swelling caused by heat shock at 41.0 or 43.0°C (Fig. 8H).
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Microfilament Reorganization in Heat-Shocked Cells
Localization of microfilaments using fluorescently labeled phalloidin was performed to determine whether heat shock of 43.0°C caused changes in microfilament morphology.
Microfilaments at 38.5°C were observed throughout the blastomere as irregularly shaped fibers (Fig. 10, A, C, and E) with a relaxed and entangled (meshlike) appearance. Microfilaments were particularly prominent in the microvilli around the periphery of blastomeres (Fig. 10E) and underneath regions of the plasma membrane where blastomeres were apposed (Fig. 10A). Heat shock of 43.0°C caused reorganization of the actin cytoskeleton (Fig. 10, B and D). In contrast with the more diffuse localization pattern in embryos at 38.5°C, the matrix of microfilaments appeared thinned in heat-shocked embryos and individual microfilaments were more distinct in appearance, with individual short fibers being prominent. While fibers in embryos cultured at 38.5°C were usually straight, with bending seen occasionally (Fig. 10E), actin filaments in the heat-shocked embryos were shorter, with a rod-like appearance, and showed breakage (Fig. 10F). In heat-shocked cells, moreover, the band of microfilaments along the boundary of blastomeres was broader and less dense as compared with control cells and the microvilli were no longer apparent (Fig. 10, A–D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alterations in cytoskeletal organization in response to heat shock have been reported in a variety of cell lines in culture [14, 15, 24]. In addition, redistribution of organelles to the perinuclear region has been observed upon heat shock [15]. The effect of heat shock on the three components of the cytoskeleton, namely microtubules, microfilaments, and intermediate filaments, is different for different cell types [25]. Moreover, the effects of heat shock on the organization of the cytoskeleton are most likely dependent on the severity of the heat shock and duration of treatment. Here it is demonstrated that the control of organellar movement caused by heat shock depends on temperature. At 41.0°C, organellar movement away from the periphery of the cell could be blocked by either microfilament or microtubule depolymerization. Such a result indicates that damage to both components of the cytoskeleton is required for organellar movement at this temperature. In contrast, at the more severe heat shock of 43.0°C, microtubule depolymerization action with rhizoxin blocked organellar movement but microfilament depolymerization had minimal effects on movement of organelles from the cell periphery. Heat shock at 43.0°C was sufficient to disrupt microfilament structure, as revealed by studies using a phalloidin fluoroprobe to localize actin. The actin rods forming during exposure to 43.0°C have previously been documented in several mouse cell lines exposed to elevated temperatures [26]. Presumably, movement of organelles occurred in embryos treated with latrunculin B because the disruption in microtubule structure at 43.0°C was so extensive as to cause movement of organelles even when microfilaments were depolymerized by latrunculin B.
The fact that paclitaxel did not exert the same effects as rhizoxin to block effects of heat shock on redistribution of organelles away from the plasma membrane (or, as discussed below, on mitochondrial swelling) implies that the movement of organelles and swelling of mitochondria induced by heat shock requires intact microtubules but does not require microtubules to be capable of depolymerization. Thus, even though elevated temperature can induce depolymerization of microtubules in cell-free suspension [27], this phenomenon does not seem to be important for disruption of the microtubule network in the heat-shocked blastomere because changes occurred in the presence of paclitaxel. Perhaps heat shock causes denaturation of microtubules in a way that alters their shape and position in the cell. Alternatively, heat shock could distort the function of molecular motors on microtubules such as dynein. Such actions of heat shock could conceivably also occur when depolymerization is inhibited by paclitaxel.
Among the effects of heat shock is an increase in the number of swollen mitochondria at both 41.0 and 43.0°C. Mitochondria are arranged along microtubules [28, 29], and intracellular distribution and appearance of mitochondria is regulated by association with microtubules [30, 31] and altered when microtubules are disrupted [28, 32, 33]. The importance of microtubules for mitochondrial positioning was illustrated in the current study by the reorganization of mitochondria into columns along the nuclear-periphery axis upon treatment with paclitaxel. The swelling of mitochondria in response to heat shock appears to require intact microtubules because this phenomenon was blocked by rhizoxin.
Cell-specific actions of heat shock on intermediate filaments probably reflect that intermediate filaments are composed of a multigene family of proteins that are expressed in cell-type-specific patterns [34]. Whether heat shock affects intermediate filaments in preimplantation embryos is unknown. Indeed, there is disagreement as to the presence of intermediate filaments in the early mammalian embryo [35]. Intermediate filaments existing as fibrous sheets of keratin filaments have been described in bovine oocytes and preimplantation embryos [36], and what appeared to be intranuclear intermediate filament bundles were observed in some of the embryos exposed to 43.0°C in the present study. Similar structures composed of cytokeratin have been described in preimplantation embryos of the hamster [37]. Perhaps the presence of this fibrillar structure in the nucleus following heat shock is indicative of disruption of intermediate filaments.
Induction of swelling in mitochondria by heat shock is a well-known consequence of heat shock [38–40] and has been recently reported to occur in bovine two-cell embryos exposed to heat shock [5]. As mentioned earlier, the finding that rhizoxin prevented this phenomenon implicates microtubules in this process. Mitochondrial swelling results from loss of membrane potential as a result of the opening of the high-conductance PTP on the inner mitochondrial membrane [41]. Opening of the PTP is typically promoted by the accumulation of excessive quantities of Ca2+ [42, 43]. The loss of membrane potential allows unselective diffusion of large molecules into the matrix [42] to cause osmotic imbalance and swelling.
There are several possible ways in which microtubules might interact with mitochondria to control opening of the PTP following heat shock. As previously mentioned, opening of the PTP is typically promoted by the accumulation of excessive quantities of Ca2+ [43]. Heat shock of 43.0°C results in increases in intracellular calcium ions due to an influx into the cytoplasm from both internal stores and the extracellular medium [44]. The ability of mitochondria to buffer calcium influx in cells is critically dependent on the microtubule-dependent spatial distribution of mitochondria [33], and changes in distribution of mitochondria caused by heat shock may increase vulnerability of this organelle to insults. Alternatively, opening of membrane pores in the mitochondria may depend on proteins linked to microtubules. One such protein is Bim (Bcl-2-interacting mediator of cell death). This Bcl-2 family protein involved in apoptosis exists either as sequestered to microtubules through binding to the dynein motor complex or in the mitochondrial membrane in association with Bcl-2 [45]. Upon an apoptotic stimuli like microtubule reorganization, Bim is released from the microtubules and translocates to the mitochondria, where it binds Bcl-2, a protein tightly associated with the voltage-dependent anion channel that represents one of the most abundant PTP components [46, 47]. Perhaps treatment with rhizoxin interferes with the ability of microtubule reorganization to induce PTP opening through Bim or another protein.
In conclusion, heat shock of two-cell bovine embryos results in changes to the microfilaments, microtubules, and possibly intermediate filaments. In addition, the damage of cytoskeletal components leads to organizational changes in the spatial organization of organelles within the cell and contributes to the swelling of mitochondria. The fact that organelles move toward a perinuclear direction implies that the secretory and absorptive functions of the embryo are impaired by heat shock. Changes in mitochondrial distribution may increase the vulnerability of this organelle to insults such as Ca2+ [33, 42] and facilitate PTP opening and swelling [42, 43]. Other large-scale pathological changes are seen in blastomeres exposed to 43.0°C, including protein and chromatin denaturation as evidenced by electron-dense material in the cytoplasm and nucleus.
A major question that arises from these conclusions is the extent to which changes in the cytoskeleton and mitochondrial function caused by heat shock at the physiologically relevant temperature of 41.0°C contributes to the inhibition of development caused by this mild heat shock. Recent unpublished results demonstrate that embryos are capable of continued cleavage after a heat shock of 41.0°C for 6 h before they block in development at the eight-cell stage. Because embryos can recover from heat shock to the point where two more cell divisions are possible, it is likely that much of the damage to the cytoskeleton caused by exposure to 41.0°C can be repaired, as described for other cells [24, 48].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Peter J. Hansen, Department of Animal Sciences, University of Florida, P.O. Box 110910, Gainesville, FL 32611. FAX: 352 392 5595; hansen{at}animal.ufl.edu
3 Current address: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018
Received: 30 October 2003.
First decision: 9 December 2003.
Accepted: 5 February 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and the nitric oxide generator, sodium nitroprusside dihydrate, on oocyte maturation and embryonic development in cattle. Am J Reprod Immunol 2003 50:263-272This article has been cited by other articles:
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Z Roth and P J Hansen Disruption of nuclear maturation and rearrangement of cytoskeletal elements in bovine oocytes exposed to heat shock during maturation Reproduction, February 1, 2005; 129(2): 235 - 244. [Abstract] [Full Text] [PDF]
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 R. R. Payton, R. Romar, P. Coy, A. M. Saxton, J. L. Lawrence, and J. L. Edwards Susceptibility of Bovine Germinal Vesicle-Stage Oocytes from Antral Follicles to Direct Effects of Heat Stress In Vitro Biol Reprod, October 1, 2004; 71(4): 1303 - 1308. [Abstract] [Full Text] [PDF]
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R. M. Rivera, G. M Dahlgren, L. A. de Castro e Paula, R. T Kennedy, and P. J Hansen Actions of thermal stress in two-cell bovine embryos: oxygen metabolism, glutathione and ATP content, and the time-course of development Reproduction, July 1, 2004; 128(1): 33 - 42. [Abstract] [Full Text] [PDF]
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