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BOR - Papers in Press, published online ahead of print March 29, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.049791
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BIOLOGY OF REPRODUCTION 75, 45–55 (2006)
DOI: 10.1095/biolreprod.105.049791
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Shear Stress Induces Preimplantation Embryo Death That Is Delayed by the Zona Pellucida and Associated with Stress-Activated Protein Kinase-Mediated Apoptosis1

Yufen Xie 3, Fangfei Wang 3, Wenjing Zhong 3, Elizabeth Puscheck 3, Hayley Shen 7, and D.A. Rappolee 2 3456   

CS Mott Center for Human Growth and Development of Ob/Gyn,3 Department of Anatomy and Cell Biology,4 Karmanos Cancer Institute,5 Institute of Environmental Health Sciences,6 Wayne State University School of Medicine, Detroit, Michigan 48201 Departments of Civil and Environmental Engineering,7 Clarkson University, Potsdam, New York 13699-5710

ABSTRACT

In this study, we discovered that embryos sense shear stress and sought to characterize the kinetics and the enzymatic mechanisms underlying induction of embryonic lethality by shear stress. Using a rotating wall vessel programmed to produce 1.2 dynes/cm2 shear stress, it was found that shear stress caused lethality within 12 h for E3.5 blastocysts. Embryos developed an approximate 100% increase in mitogen-activated protein kinase 8/9 (formerly known as stress-activated protein kinase/junC kinase 1/2) phosphorylation by 6 h of shear stress that further increased to approximately 350% by 12 h. Terminal deoxynucleotidyltransferase dUTP nick end labeling/apoptosis was at baseline levels at 6 h and increased to approximately 500% of baseline at 12 h, when irreversible commitment to death occurred. A mitogen-activated protein kinase 8/9 phosphorylation inhibitor, D-JNKI1, was able to inhibit over 50% of the apoptosis, suggesting a causal role for mitogen-activated protein kinase 8/9 phosphorylation in the shear stress-induced lethality. The E2.5 (compacted eight-cell/early morula stage) embryo was more sensitive to shear stress than the E3.5 (early blastocyst stage) embryo. Additionally, zona pellucida removal significantly accelerated shear stress-induced lethality while having no lethal effect on embryos in the static control. In conclusion, preimplantation embryos sense shear stress, chronic shear stress is lethal, and the zona pellucida lessens the lethal and sublethal effects of shear stress. Embryos in vivo would not experience as high a sustained velocity or shear stress as induced experimentally here. Lower shear stresses might induce sufficient mitogen-activated protein kinase 8/9 phosphorylation that would slow growth or cause premature differentiation if the zona pellucida were not intact.

apoptosis, embryo, kinases, signal transduction, stress

INTRODUCTION

Shear stress (SS) caused by the movement of fluid over the surface of cells was first reported in endothelial cells [1, 2]. It has been reported almost exclusively in cell types that experience laminar flow over their surfaces, such as endothelial cells, or in load-bearing cells, such as osteoblasts [3–6]. However, there have been reports of SS in other cell types involved with load bearing (e.g., chondrocytes and smooth muscle cells [7, 8]). In a model for placental transdifferentiation to endothelial cells [9], trophoblasts polarize their cytoskeleton, as do endothelial cells, in response to SS. But signaling and lethality were not investigated.

Shear stress enters the cell through not fully characterized cell surface receptors and influences nuclear decision making by phosphorylating transcription factors [10]. It induces cytoskeletal remodeling [2], activation of cytoplasmic-to-nuclear signaling cascades that include mitogen-activated protein kinase/extracellular receptor kinase (MAPK1/3, formerly known as MAPK/ERK1/2) and stress-activated protein kinase/junC kinase (MAPK8/9, formerly known as SAPK/JNK1/2), and activation of transcription factors such as FOS (formerly known as c-FOS), JUN (formerly known as c-JUN), and TRP53 (formerly known as p53) [5, 8, 11, 12]. Biological responses to chronic SS include increased adhesion [3, 9, 13], differentiation [3], enhancement of apoptosis [14, 15], inhibition of apoptosis [16–18], and growth arrest [5].

Shear stress has not been studied in embryos, but preimplantation embryos may experience fluid flow. After ovulation, oocytes are fertilized in the first segment of the oviduct. The embryos then traverse the oviduct under the propulsion of cilia and smooth muscle-mediated peristalsis and traverse the uterus solely by peristalsis [19]. In addition, in another study, we found that microgravity simulation (MGS) induced phosphorylated MAPK 8/9 and was lethal to preimplantation embryos (E2.5; unpublished results). The kinetics of lethality suggested that embryos were most sensitive at the late-eight-cell/early-morula stage, 2.5 days after fertilization. However, rotational controls that produce inconsequential MGS develop lethality at a slower rate than MGS. Therefore, rotation in the microgravity simulator might generate sufficient SS to cause lethality.

Stress-activated protein kinase/jun kinase (MAPK8/9) is induced in models of shear stress. Mapk8/9 mRNA and MAPK8/9 protein are expressed in preimplantation mouse embryos and trophoblast stem cells [20, 21]. MAPK8/9 mediates homeostatic but not developmentally essential responses to stress in embryos cultured in optimal media [22, 23]. The level of MAPK8/9 Thr183/Tyr185 phosphorylation is negatively correlated with the rate of development [21], and the activation of MAPK8/9 by an upstream cascade of kinases is marked by dual phosphorylation at Thr183/Tyr185 by MAPK2K4/MAPK2K7 (formerly known as MKK4/7) [24] that leads to an opening of ATP and substrate binding sites. In optimal media such as KSOM+AA [25–27], two developmental events are accelerated when MAPK8/9 inhibitors are present during embryo culture from the late two-cell stage (E1.5) for 72 h [23]. Taken together, these data suggest that phosphorylated MAPK8/9 are good markers of stress during embryo perturbation experiments and that MAPK8/9 inhibitors can be used to test for attenuation of stress during perturbation.

In this report, we show that preimplantation embryos respond to SS with a rapid induction of FOS and a slower induction of MAPK8/9 that precedes and is causal for DNTT (formerly known as terminal deoxynucleotidyltransferase) dUTP nick end labeling (TUNEL)/apoptosis and lethality. Additionally, we speculate that a novel function for the zona pellucida may be the protection of the embryo from SS during movement through the oviduct and uterus.

MATERIALS AND METHODS

Media and Embryo Culture Manipulation

KSOM+AA (supplemented with amino acids) [28] (Specialty Media, Phillipsburg, NJ) and mineral oil (Sigma Chemical Co., St. Louis, MO) were equilibrated overnight at 37°C in 5% CO2 before the embryo culture. For inhibition of MAPK8/9, 1 µM D-JNKl1 (Alexis, San Diego, CA) was used. D-JNKl1 is a fusion protein of the TAT delivery peptide from HIV and the oligopeptide representing the interaction domain between MAPK8/9 and the MAPK8/9 interacting protein MAPK8IP1 (formerly known as JNK interacting protein [JIP]-1). MAPK8IP1 is a naturally occurring inhibitor of MAPK8/9 nuclear localization and activity [29–31]. In a previous study, both D-JNKl1 and a second MAPK8/9 inhibitor (SP600125) were used to increase embryonic development in KSOM+AA, but D-JNKl1 can be used at 10–100-fold lower concentrations than SP600125 and does not yet have reports of nonspecific inhibition of other kinases as does SP600125 [23 and citations therein].

Hyperosmolarity Dose-Dependent and Time-Dependent Effects on Embryo Lethality

To estimate the potency of lethality induced by SS, we established the time and dose dependence of embryo lethality using hyperosmolarity mediated by sorbitol, a common method for activating apoptosis and stress enzymes in somatic cells [32, 33]. Individual embryos were cultured in 5 µl KSOM+AA microdroplets under oil without or with sorbitol at 50, 200, 600, and 1000 mM. Phase micrographs were taken at 0, 0.5, 1, 3, 4, 24, and 48 h of culture. Final osmolarity was measured by crystallizing KSOM+AA/sorbitol media samples and assaying on a model 3W2 osmometer per manufacturer's instructions (Advanced Instruments, Inc., Needham Heights, MA). The increase in osmolarity in KSOM+AA media due to sorbitol addition was (sorbitol mM/mOs = milliosmolar) none/239 mOs, 50 mM/298 mOs, 200 mM/490 mOs, 600 mM/860 mOs, and 1000 mM/1403 mOs.

Collection of Mouse Embryos

Techniques for obtaining mouse embryos were described previously [34]. Female MF-1 mice (4–5 wk old; Harlan Sprague Dawley, Indianapolis, IN) were injected intraperitoneally with 7.5 IU of pregnant eCG (Sigma), followed by an injection of 7.5 IU of human chorionic gonadotropin (Sigma) 44–48 h later. After the second injection, females were housed overnight with C57BL/6J xSJL/J F1 hybrid males (Jackson Laboratories, Bar Harbor, ME). Noon of the day following coitus was considered Day E0.5. Embryos were obtained at the compacted eight-cell/early morula (E2.5) and late-morula/early-blastocyst (E3.5) stages. Embryos were flushed out from the oviduct (E1.5 and early E2.5) or uterus (late E2.5 or E3.5) with cold M2 medium (Specialty Media) with minimal handling to minimize stress response during handling. Embryos were washed twice in KSOMaa medium to remove the M2 medium. Embryos were allowed to incubate in KSOMaa medium for 2 h before the start of the treatment to recover from the stress from flushing. Animal use protocols were approved by the Wayne State University Animal Investigation Committee.

Indirect Immunocytochemistry and Nuclear Staining

For immunocytochemical analysis, embryos were fixed for 30 min in 2% fresh paraformaldehyde (pH 7.4) in phosphate-buffered saline (PBS), quenched with 0.1 M glycine, and permeabilized for 10 min with 0.1% Triton X-100. Embryos were stained with rabbit polyclonal and monoclonal anti-human phosphorylation-specific MAPK8/9 (Thr183/Tyr185) (CS9251), and rabbit polyclonal anti-human all forms FOS proto-oncogene (SC52 and UBI-06–341) (CS = Cell Signaling Technology, Inc., Beverly, MA; SC = Santa Cruz Biotechnology, Santa Cruz, CA) (diluted at 1:100 in PBS-Tween with 10% fetal calf serum [FCS]). In our terminology, "all forms" antibodies detect both phosphorylated and unphosphorylated proteins. The primary antibody was followed with a biotinylated anti-rabbit IgG secondary antibody, proteins were visualized with streptavidin-fluorescein isothiocyanate (FITC), nuclei were counterstained with Hoechst 33258 (10 µg/ml), and photomicrography was done with a Leica DM IRE2 epifluorescence microscope as previously described [29–31]. Primary and secondary antibodies were incubated for 1 h, and three wash steps between incubations lasted for at least 15 min. Specificity controls included incubation with no first antibody or incubation of first antibody with excess immunizing antigen. Photomicrographs were formatted using Adobe Photoshop 6.0 (San Jose, CA). FITC-conjugated intensity measurement and comparison were done with SimplePCI software (Compix Imaging Systems, Cranberry Township, PA). Generally, photomicrography was optimized for each antibody (FOS and phosphorylated MAPK8/9) independently by setting exposure time so that no antibody control gave less than 10 units of arbitrary intensity; the experimental groups ranged from approximately 20 to 100 arbitrary units. Intensities were in linear range, and no intensity was saturated (maximal intensity = 255). Stimulation indices for fluorescence intensity were similar to those measured by scanning densitometry of similar samples by Western blot analysis [21]. Because of the inherent differences in antibody-antigen affinity and optimization of exposure time for each antibody, no comparisons between antigens were intended in this study.

Western Blotting

Embryos were washed twice with 10 µl ice-cold PBS; 10 µl 2x cell lysis buffer (Cell Signaling) plus phosphatase inhibitor cocktail (PIC1; Sigma) and (PIC2; Sigma) were added together, and embryos and 10 µl ice-cold PBS were pipetted into 2x cell lysis buffer to make a 1x lysis buffer. Each group of 150 embryos was incubated for 20 min on ice. The lysates were centrifuged for 10 min, and the supernatant was stored at –80°C. The proteins in 20 µg of whole-cell extracts were separated by electrophoresis on a 10% SDS-PAGE gel using a Hoefer Mighty Small II SE 250 (San Francisco, CA) apparatus and then transferred to ECL Hybond nitrocellulose membranes (Amersham) at 15 V for 30 min using a Bio-Rad semidry transfer cell. The membranes were blocked overnight with 5% nonfat milk in Tris-buffered saline-Tween20 (TTBS) and blotted with the specified primary antibodies for 1 h followed by three washes in TTBS and then incubated in horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h followed by extensive wash with TTBS. Primary and secondary antibodies were diluted in 1% nonfat milk/TTBS. The protein bands were visualized using the enhanced chemiluminescence (ECL) assay system (Amersham) [21, 29].

SS Protocol and Calculations

In static high-aspect ratio vessel (HARV), embryos progress at the same rate as in a microdroplet culture under oil in plastic dishes (data not shown), suggesting that the apparatus does not adversely affect embryos. Thus, to shear stress preimplantation mouse embryos, we used a Synthecon (Houston, TX) rotating wall vessel apparatus (Rotary Cell Culture System, RCCS1) with discus-shaped 10-ml HARV vessels designed to simulate microgravity and used to do so in embryos (NASA, http://microgravity.msfc.nasa.gov; unpublished results; Fig. 1A) [35].


Figure 1
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  		FIG. 1. SS was done using a rotating wall vessel in the "Ferris wheel orientation." A) Volumes of oil and KSOM+AA embryo media and number of embryos were the same in SS (a) and SC (b). B) Schematics of SS force with the stationary aqueous embryo media microdroplet (gray circle) falling through oil that filled the counterclockwise-rotating HARV (large circle, a) and the diagram of the inner clockwise (left, toward the center of the microdroplet) and outer counterclockwise (right, toward the outer rim of the HARV) streamlines inside the aqueous microdroplet (b). Laminar flow equations (Supplement 1, available online at http://www.biolreprod.org) predicted two counterrotating spheres inside the microdroplet. Videography of the microdroplet confirmed embryo movement and gave velocity data that, after being plugged into the equations, yielded an SS calculated at 1.2 dynes/cm2.

Experiments consisted of SS and static control (SC) groups. For SS, the RCCS1 was placed in the "Ferris wheel orientation" (rotational axis horizontal) and the HARV filled with pre-equilibrated mineral oil. Microdroplets of 50 µl media containing 50 mouse embryos were placed into the oil, and air bubbles were removed. The HARV was rotated at 7.5 rpm. The SC groups were standard and consisted of 50 embryos in 50 µl under oil, as done previously [34, 36–38].

The generation of shear forces on an embryo is a three-step process. First, the walls of the HARV impart force on the oil, quickly building up to a "solid body rotation" where the angular velocity of the oil is equal to that of the HARV.

Second, the denser aqueous ({rho}droplet = 1 g/m3) microdroplet falls through the lighter oil (Fig. 1B; {rho}oil = 0.84 g/m3). At a microdroplet size of about 50 µl, it is in stable suspension within the moving oil. If smaller, it rotates with the oil, resulting in a force balance simulating microgravity. If larger, it falls to the bottom of the HARV and bounces. At the stable suspension size, the microdroplet remains in one position, constantly falling through the oil at a velocity equal to that of the oil moving upward around it. As a function of this velocity, the viscosity differences at the oil-aqueous interface, and the drag of the surfaces of the oil and media, a force is imparted into the microdroplet that causes the media to form internal circulations.

Third, inside the microdroplet, the denser embryos fall through the less dense media. However, the internal circulation of the media deflects the falling embryos into orbits along the flow fields inside the microdroplet. These flow fields, which are related to the internal circulations of the media, can be calculated and the embryo orbits estimated on the basis of laminar flow equations (see the following equation and Supplement 1, available online at http://www.biolreprod.org).

The flow fields inside and around the microdroplet containing the embryos have been solved analytically using laminar flow equations [39 and Supplement 1]. Figure 1B shows the microdroplet in the rotating HARV (a), and Figure 1B shows the streamlines of the flow inside and around the microdroplet (b). A Zeiss dissecting microscope mounted with a Javelin B/W CCD (Los Angeles, CA) was used to record and confirm the embryo orbits, their velocities, and the two counterrotating flow spheres in the microdroplet.

The SS magnitude can be estimated by describing the relative motion and density of the embryos orbiting an aqueous media microdroplet. For a free-falling object such as the embryo inside the less dense media, the force balance requires the gravity force on the embryo to be canceled, with the buoyancy of the media and the drag force resulting from the SS. The rate of embryos falling through KSOM+AA, which is of known density, defines the density of the embryos relative to the aqueous media. For example, if embryo density equals 1.5 g/cm3, then the embryo will drop 2 mm in 3 sec; a drop of 6 mm in 3 sec requires a density of 2.45 g/cm3. Since the embryo density is greater than the media, it will drop out of the flow field and sink to the bottom of the microdroplet, unless the flow velocity in the microdroplet is higher than the terminal velocity of the falling embryo.

The embryos are observed dropping in and out of different orbits because of the density difference induced by the velocity of the fall. In a series of four equations, we define 1) the acceleration of the microdroplet due to the net effect of its weight relative to the buoyancy of the oil and the shear-induced drag force, 2) the displacement of a falling object in a lighter fluid, 3) the orbiting velocity of the internal circulation of a droplet falling through another fluid, and 4) the acceleration of the embryo subject to its weight, buoyancy, and the drag force influenced by its orbital motion (see Supplement 1, available online at http://www.biolreprod.org).

Finally, we solve for SS. In the following equation, the free-fall velocity of the embryo is 0.6/3 cm/sec as measured:

Formula

Therefore, SS is calculated at 1.2 dynes/cm2, and the maximal velocity is observed to be 261 ± 32.2 µm/sec (data not shown).

Embryo Culture and Evaluation

To study the effect of SS, preimplantation mouse embryos, isolated at E2.5 and E3.5, were divided into two groups: SS and SC. Static control is a culture control for incubator/media conditions without motion. After 6–24 h of culture rotation, E3.5 embryos were examined under a microscope (Leica DM IRE2) for developmental progression (the number of embryos developed to the cavitated blastocyst and hatched blastocyst stages) and for visual signs of morbidity and death (embryos with collapsed blastocyst cavities, retraction from the zona pellucida, opaqueness, and fragmentation of cells). Morbid embryos met one or two of these criteria or several partial criteria, and dead embryos met all criteria. In addition, embryos were cultured an additional 12–24 h in an SC condition to determine if morbidity and death assigned by visual assays actually resulted in death. As in prior studies, after an SS or SC culture, embryos were subjected to an ICC with antiphosphorylated MAPK8/9 and anti-MAPK8/9 to determine the stress during the past 24 h of culture on a molecular level using previously described criteria for evaluating blastocyst formation and hatching [20, 21].

DNTT dUTP nTUNEL Assays

The embryos were washed and fixed in 2% (v/v) paraformaldehyde/PBS solution for 30 min at room temperature. For membrane permeabilization, the embryos were incubated in 0.1% Triton X-100 in 0.1% citrate solution for 10 min. A TUNEL assay was then used to assess the presence of apoptotic cells (DeadEnd Fluorometric TUNEL System; Promega, Madison, WI).

Fixed embryos were incubated in a TUNEL reaction medium containing an equilibration buffer, nucleotide mix, and rTdT enzyme at 37°C for 1 h and then washed and transferred into 2 mg/ml of DAPI (49,6-diamidine-29-penylindole dihydrochloride; Roche). The fraction of TUNEL-positive cells was quantitated in embryos by visually inspecting them using the Z-axis control of an epifluorescent microscope (Leica DM IRE2; Germany). The criteria for assigning a positive status were the colocalization of TUNEL product around a single DAPI-stained nucleus, above the background level of TUNEL staining in normal, and unperturbed static culture embryos in areas that lacked any elevated TUNEL staining.

Statistical Analysis

Single sets of micrographs and graphic data were representative data from three replicate experiments with similar outcomes and data were presented as mean ± SD. The statistical significance of differences between treated samples was analyzed by Student t-test (for continuous data with more than two groups), chi-square (used to determine the differences in groups measured in percentage), or by one-way ANOVA (when there was one variable and more than two groups). Further analysis, consisting of least significant difference post hoc tests, was done when the analysis of paired data by ANOVA showed a significant difference (SPSS 11.0). Two levels of significance were determined: P ≥ 0.05 was insignificant and P < 0.05 was significant. All cause-and-effect experiments and immunofluorescence studies were repeated three times, and Westerns were repeated twice.

RESULTS

Time-Dependent Generation of SS Response Leads to Morbidity and Lethality; E2.5 Embryos Are More Sensitive to SS Than E3.5 Embryos

When a rotational control in a previous study of MGS also created delayed lethality, we concluded that rotation in the microgravity simulator might generate sufficient SS to cause lethality. The Synthecon RCCS1 rotating wall vessel was modified to produce shear force to embryos of 1.2 dynes/cm2 with no MGS.

We tested for the time dependence of SS for several parameters of embryo health. Embryos were subjected to SS for 12 h and then assayed for the fraction of unhealthy embryos at the end of the perturbation culture. We assayed again after transfer to a static recovery culture for an additional 24 h (Fig. 2A). At the end of 12 h of SS, embryos were clearly morbid, and no living embryos were recovered after 24 h in the static culture (compare Fig. 2A, panels c, e). Whereas for the SC culture, very few embryos were morbid at the end of the initial 12 h or after the additional 24 h (Fig. 2A, panels d, f). This suggests that SS has a lethality at 12 h that is not reversible, even after 24 h of recovery culture.


Figure 2
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  		FIG. 2. Lethality in SS is time dependent. A) E3.5 embryos were stimulated by 12 h of SS or SC, then transferred to static recovery culture for 24 h. a, b Show embryos before treatment; c, d show embryos after 12 h of treatment; and e, f show embryos after an additional 24-h recovery period. A 50-µm bar is shown in (f). Magnification x200. B) The line graph shows the quantitative measurements of lethality. a, Significant differences compared with SC at 6-h time point (P < 0.05). b, Significant differences compared with SC at 10 h (P < 0.05). d, Significant differences compared with SC at 12 h (P < 0.05). Sample sizes for SS at 6, 10, and 12 h were 29, 35, and 25 embryos, respectively. Sample sizes for SC at 6, 10, and 12 h were 35, 41, and 34 embryos, respectively. * Indicates lethality of E2.5 embryo at 10 h. C) Line graph shows quantitative measurements of cell number (mean ± SD). a, Significant differences compared with SC at 8 h (P < 0.05). b, Significant differences compared with SC at 10 h (P < 0.05). Sample sizes for SS at 6, 10, and 12 h were 40, 34, and 20 embryos, respectively. Sample sizes for SC were 36, 26, and 22 embryos, respectively.

In experiments similar to these, we tested for the rapidity of the onset of lethality before 12 h. At 10 h, we found that about 44% of the embryos in SS were dead or would die in recovery and that SC had less morbidity and death. At 6 h, there was no death in SC groups, but about 13% of SS embryos were dead (Fig. 2B). This suggests that SS estimated to be 1.2 dynes/cm2 creates a lethality that increases from relatively low levels at 6 h to completion at 12 h.

Since we observed more rapid effects of MGS in compacted eight-cell/early morula (E2.5) than late morula/early blastocysts (E3.5) (unpublished results), we also tested E2.5 embryos for effects of SS. We found that SS was completely lethal for E2.5 embryos by 10 h of rotation (Fig. 2B), indicating that E2.5 embryos are more sensitive to SS than E3.5 embryos since at 10 h E3.5 embryos had only 43% embryo death and 100% of E2.5 embryos were dead.

We also tested for the total cell number as a function of time during SS. Compared with SC, E3.5 embryos had statistically fewer cells at 10 h (52.6 compared to 77.1 per embryo) and 12 h (35.5 compared to 79.9 per embryo) for SS (t-test; P < 0.01) (Fig. 2C). However, it should be noted that cell counts are difficult to perform when the embryos exceed 80–90 cells, and nuclei were counted conservatively. Therefore, actual cell numbers at 12 h may be higher than counted. In conclusion, these data suggest a net cell loss during SS that may involve apoptosis.

Shear-Stressed E3.5 Embryos Develop Elevated MAPK8/9 Phosphorylation That Precedes Death

Since we anticipated that the decline in health of the embryos might be preceded by the activation of stress enzymes, we tested for markers of stress signaling inside the embryos. An increase was found in MAPK8/9 phosphorylation in late morula/early blastocysts (E3.5) after 12 h of SS (Fig. 3A), when 100% of embryos are irreversibly committed to death. At 10 h, phosphorylated MAPK8/9 bands of predicted 46 and 54 kDa were induced in SS embryos, where actin controls showed even loading of total embryo protein, as detected by Western blot analysis. In similar experiments, we measured average fluorescence intensity of phosphorylated MAPK8/9 at three time points (6, 8, and 10 h) preceding complete lethality. Increasing levels of MAPK8/9 phosphorylation were detected in shear-stressed embryos from 6 to 12 h (Fig. 3B), indicating that SS activates phosphorylated MAPK8/9 and that the rise in MAPK8/9 phosphorylation precedes the lethality of SS embryos at 12 h.


Figure 3
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  		FIG. 3. SS perturbed E3.5 embryos express higher levels of phosphorylated MAPK8/9 than SC. A) E3.5 embryos were subject to 10 h of SS and SC and tested for levels of phosphorylated MAPK8/9 (a and b, respectively) or no antibody (c). A 50-µm bar is shown in (a). Magnification x200. Western blot (d) showed higher levels of phosphorylated MAPK8/9 in E3.5 embryos stimulated by SS for 10 h (lane 1) than SC (lane 2). Molecular weights of markers (not shown) were 46.5 and 57 kDa, suggesting that MAPK8/9 were approximately 46 and 54 kDa and that ACTB (formerly known as actin beta) was 45 kDa as expected. B) The line graph shows the quantitative immunofluorescence measurements of SS and SC embryos at 6–12 h that includes the 10-h time point shown in A (mean ± SD). a, Significant difference compared with SC at 6 h (P < 0.05). b, Significant difference compared with SC at 8 h (P < 0.05). c, Significant difference compared with SC at 10 h (P < 0.05). d, Significant differences compared with SC at 12 h (P < 0.05). Sample sizes for SS at 6, 8, 10, and 12 h were 18, 28, 25, and 20 embryos, respectively. Sample sizes for SC at 6, 8, 10, and 12 h were 21, 33, 25, and 23 embryos, respectively.

Time-Dependent Generation of SS Response Leads to Apoptosis/TUNEL That Is MAPK8/9 Dependent

Since elevated MAPK8/9 can lead to a lethal phenotype in endothelial cells [33], we tested for the time dependence of the generation of apoptosis by testing for terminal transferase-mediated dUTP TUNEL [40, 41]. TUNEL is an accepted way to test for apoptosis in preimplantation mammalian embryos [42, 43]. After 10 h, embryos perturbed by SS displayed an increased TUNEL (Fig. 4A). TUNEL-positive cell numbers showed a time-dependent increase in E3.5 blastocysts during SS (Fig. 4B). The TUNEL-positive time dependence paralleled and followed the time course for phosphorylated MAPK8/9 (phosphorylated MAPK8/9 at 6, 10, and 12 h was 1.8-, 2.2-, and 3-fold compared with static control, respectively, whereas the TUNEL at 6, 10, and 12 h was 1.1-, 1.8-, and 5-fold compared with static control, respectively). At 6 h, phosphorylated MAPK8/9 in SS was already above SC levels, but TUNEL was not.


Figure 4
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  		FIG. 4. SS-perturbed E3.5 embryos express higher numbers of TUNEL-positive cells than SC. A) Embryos were subject to 12 h of SS (a) and SC (b) and assayed for TUNEL by TdT binding. (c) Represents the no TdT control. A 50-µm bar is shown in (a). Magnification x100. B) Quantitative measurements of percentage of TUNEL-positive cells at 6–12 h, including the 12-h time point in A (mean ± SD). a, Significant differences compared with SC at 10 h (P < 0.05). b, Significant differences compared with SC at 12 h (P < 0.05). Sample sizes for SS at 6, 10, and 12 h were 40, 34, and 20 embryos, respectively. Sample sizes for SC were 36, 26, and 22 embryos, respectively.

To test for the role of MAPK8/9 in TUNEL induction by SS, we stressed embryos in the presence of the MAPK8/9 inhibitor D-JNKI1. D-JNKI1 has been used in somatic cells [30, 31] and in placental stem cells and preimplantation embryos [23] to reduce the induction of MAPK8/9 activities, such as Jun phosphorylation, apoptosis, and cell cycle arrest. D-JNKI1 blocked SS-induced TUNEL increases at 12 h of SS (Fig. 5A) and reduced TUNEL-positive cells by about 50% (Fig. 5B). There also was a significant increase in cell number in the MAPK8/9-inhibited group (57 ± 5.1 compared to 38.9 ± 3.4 per embryo, P < 0.05). This experiment suggests that MAPK8/9 precedes TUNEL/apoptosis and contributes to TUNEL induction.


Figure 5
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  		FIG. 5. E3.5 embryos were protected from SS-induced TUNEL induction and death by the MAPK8/9 inhibitor, D-JNKI1. A) E3.5 embryos were subject to 10 h of perturbation by SS without (a) or with (b) 1 µM MAPK8/9 inhibitor D-JNKI1 or in SC (c) or with SC but no TdT (d). A 50-µm bar is shown in (a). Magnification x100. B) The histograms at the bottom show the relative numbers of TUNEL-positive cells (left histogram) and of total cells per embryo (right histogram) immediately after SS with or without treatment with D-JNKI1 or after SC (mean ± SD). a, Significant differences compared with SC (P < 0.05). b, Significant differences compared with DJNKI1 (P < 0.05). c, Significant differences compared with SC (P < 0.05). Sample sizes for both histograms for SS, SS+D-JNKl1, and SC were 15, 19, and 19 embryos, respectively.

FOS Protein Is Induced in Outer Trophectodermal Cells of the Blastocyst, Indicating SS Effects

Shear stress activates a rapid biphasic de novo synthesis of Fos proto-oncogene mRNA and FOS protein in endothelial and bone cells [6, 12, 44]. We tested whether the SS and SC induced FOS protein in E3.5 embryos after 30 min of perturbation. E3.5 embryos had elevated levels of FOS protein in the outer trophectodermal cells of the blastocyst but not in the inner embryonic cells 0.5 h after SS began (Fig. 6A), and SC induced an even lesser response or no response at all.


Figure 6
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  		FIG. 6. SS-perturbed E3.5 embryos express higher fluorescence intensity of FOS proto-oncogene protein than SC. A) E3.5 embryos were subject to 0.5 h of SS (a) and SC (b), and FOS expression was detected. A 50-µm bar is shown in (a). Magnification x200. B) The histogram at the bottom shows the quantitative measurements of A (mean ± SD). The black column represents the intensity of outer trophectodermal cells (TE), and the white column represents the intensity of inner cell mass (ICM). a, Significant differences compared intensity of TEC in SS with SC (P < 0.05). b, Significant differences compared intensity of TE with intensity of ICM in SS (P < 0.05). Red immunofluorescence shows actin (not shown individually). Sample sizes for the SC and SS groups were both 25 embryos.

This experiment was repeated three times with two different antibodies to FOS proto-oncogene with similar results, although in one experiment some FOS induction was apparent in the inner embryonic cells as well as the outer trophectodermal cells of the blastocyst. These data are consistent with the SS-specific generation of a rapid FOS protein induction by the outer trophectodermal cells of the blastocyst. FOS fluorescence intensity increased about 3-fold over SC during 0.5 h of SS (Fig. 6B), suggesting that SS-induced genes in preimplantation embryos include those seen in other models of SS in somatic cells.

Induction of Embryo Death Is Delayed by the Presence of the Zona Pellucida; the Toxicity of SS-Induced Lethality Is Greater Than High Doses of Hyperosmolar Stress

It was not anticipated that preimplantation embryos would sense SS. Thus, several questions arose from the shear stress data: Might preimplantation embryos moving through the oviduct and uterus sense SS, and might the zona pellucida reduce this shear? And how potent is SS-induced lethality relative to other forms of stress?

To test whether the zona pellucida might protect the embryo from SS, we removed the zona pellucida with pronase treatment and placed treated and untreated embryos in SS or SC conditions for 8 h, then transferred all embryos to SC for recovery for 12 h (Fig. 7A). An 8-h treatment was chosen because death had not occurred by this time (about 30% lethality and only minimal cell number loss interpolated from Fig. 2, B and C, respectively). Although some collapse of blastocyst cavity and membrane ruffling was seen in the trophectodermal epithelium immediately after pronase treatment, this largely disappeared after 8 h of SC. After an additional 12 h of SC culture, pronase-treated embryos were indistinguishable from untreated embryos. However, the embryos treated with pronase and subjected to 8 h of SS were mostly collapsed, opaque, and fragmented, and none were alive following the recovery culture. But 8 h of SS caused less than half (47% in this experiment) of the lethality of embryos with the zona pellucida. Therefore, removing the zona pellucida led to 100% embryo death at 8 h, well before the 100% death at 12 h for zona-intact embryos (compare Fig. 7A and Fig. 2A). This indicates that the zona pellucida can protect SS-induced lethality in vitro, and it may also offer protection in vivo.


Figure 7
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  		FIG. 7. Removal of the zona pellucida accelerates lethality caused by SS but has no effect on embryos in SC. A) E3.5 embryos were micrographed at time zero without pronase treatment (a, c; T0) or immediately after pronase treatment (b, d; T0), after SS for 8 h (e, f; T8) or SC for 8 h (g, h; T8) and then after 12 h in recovery SC (i, j, k, l). A 50-µm bar is shown in (l). Magnification x200. B) E3.5 embryos were cultured individually in microdroplets under oil with 0 mM sorbitol (left series) or 1000 mM (right series) and micrographed at 0 (not shown), 0.5, 1, 3, 4, 24, and 48 h. Magnification x200. All sample sizes were 15 embryos. C) The line graph of the results from A and B show (and including several other doses of sorbitol not represented in B) the lethality resulting from SS with or without the zona pellucida and the lethality caused by different doses of hyperosmolar sorbitol. a, Significant differences compared with embryo with zona pellucida (P < 0.05). Sample sizes of zona + and the zona – groups were 25 and 26 embryos, respectively.

To test for the relative toxicity of the SS protocol, we performed a dose response and time course using sorbitol to produce hyperosmolar stress. In other research, we found that seven embryo culture media created a range of stress equivalent to <25 mM to >200 mM sorbitol [21]. These media conditions, or a sorbitol dose range between 25 and 400 mM, decreased embryo progression and increased TUNEL cells/embryos but did not immediately kill embryos (within 24 h of culture). Therefore, embryos were cultured in individual 5 µl KSOM+AA microdrops under oil with 0, 50, 200, 600, and 1000 mM sorbitol (creating respective osmolarity of 239, 298, 490, 860, and 1403 mOs) and micrographed at 0, 0.5, 1, 3, 4, 24, and 48 h (representative data at 0 and 1000 mM; Fig. 7B; other sorbitol dose data not shown).

The fraction of dead embryos (opaque, collapsed, or fragmented) for a zona pellucida removal experiment and a sorbitol dose-response time course experiment were graphed together to compare the relative toxicity of SS on embryos with or without a zona pellucida or with 1 M sorbitol (1403 mOs) hyperosmolar stress, respectively (Fig. 7C). The data suggest that SS is very potent, inducing 42% or 100% of the embryos to die by 8 h (with or without zona pellucida, respectively), whereas very high hyperosmolarity at 1000 mM sorbitol requires >24 h (and perhaps as much as 48 h) to kill 100% of the embryos. In these tests, removal of the embryos from 1000 mM sorbitol showed they were still committed to lethality by 12 h (data not shown), indicating that removal of the zona pellucida makes the embryos more than twice as susceptible to SS-induced lethality at 8 h of treatment and that SS is more potent than a very high dose of hyperosmolar stress.

DISCUSSION

Summary

The results presented here suggest that SS is a potent inducer of embryonic lethality and that the zona pellucida protects the embryo from SS-induced lethality. MAPK8/9 phosphorylation is induced by SS preceding TUNEL induction that is associated with embryo death. This is the first report stating that trophectodermal cells of the blastocyst in whole embryos sense SS and that SS may lead directly to apoptosis.

SS Induction Is Indicated by FOS Protein Induction, and Death Is More Rapid in Embryos Without a Zona Pellucida

It was previously reported that isolated cultured macaque placental trophoblasts may sense SS [9]. But in that report, placental trophoblasts were derived from a postimplantation placenta, later in development. The trophoblasts may have been undergoing a known transdifferentiation to endothelial cells, where the placenta invades the maternal efferent vascular tree and replaces the maternal endothelial cells [45]. Our data suggest that the earlier preimplantation placental trophoblasts sense SS, as indicated by rapid cell-type-specific induction of FOS protein.

The level of Fos mRNA is nearly zero under normal conditions in preimplantation embryos ex vivo [46] and in TS cells (Liu et al., unpublished data), as is the normal level of FOS protein. Interestingly, the induced FOS protein is not uniquely nuclear, although FOS is a transcription factor and is normally detected in the nucleus after induction by SS [6, 44, 47]. It is likely that FOS in embryos has functions via participation in AP1 (formerly known as activator protein [AP]-1) transcription factor heterodimers, as in the endothelial cell response to SS [14]. MAPK8/9 is induced by SS and in turn induces activation of AP1 (particularly FOS and JUNB, formerly known as Jun-B) that leads to pejorative AP1 function and apoptosis [48, 49]. The increased susceptibility of embryos without a zona pellucida and the induction of FOS protein in outside cells of the embryo suggest that the outer trophectodermal cells of the embryo sense SS.

Hypothetical Mechanisms for Transducing Shear Through the Zona Pellucida and into the Trophectoderm Cells

There are three general ways that SS could be communicated through the zona pellucida. First, fluid percolates directly through the protein lattice of the zona pellucida or through the canaliculi remaining from the cumulus cells. Although oviduct-specific proteins reach the perivitelline space [19, 50], confirming this mode of transport, the fluid speed is likely to be so slow that significant shear is not created.

Second, stress might be transduced mechanically through the zona pellucida and act on the perivitelline space or on the surface of the trophectoderm. If shear were acting between the inner zona pellucida surface and a fluid perivitelline space, embryo spinning would be expected. This has been observed by only one group [51]. Others, using similar methodologies, have not detected embryo spinning (Roger Pedersen, Richard Gardner, and Ann Sutherland, personal communication). However, sperm movement through the zona pellucida is consistent with a fluid perivitelline space (H. Croxatto, personal communication).

An alternate hypothesis is that the zona pellucida is a solid-state matrix and develops an angular velocity communicating directly to the trophectodermal apical membrane. Such a state of the perivitelline space is supported by some evidence [52].

Third, projections of the trophectoderm through the zona pellucida are known in six species of mammals, including mice [53], and exist for about 6 h around the period of hatching in the guinea pig [54, 55].

Of these three general mechanisms, one or more may carry the SS signal to the trophectoderm cells. Interestingly, SS acting directly on the trophectoderm projecting through the zona pellucida at implantation might induce MAPK8/9 and other stress enzymes that activate maternal recognition-of-pregnancy proteins arising after implantation. We have observed maternal recognition proteins and mRNA to be induced by stress by MAPK8/9-dependent and -independent means in TS cells.

Considerations of Velocity and SS During Experimental and In Vivo Conditions

To understand the relevance of SS generated in vitro, it is important to translate the experimental velocity and SS observations to conditions occurring in vivo. We have calculated the average velocity (7.2 ± 0.08 µm/min) in the MF-1 strain by simply dividing the average length of the oviduct and uterus (mean ± SD of 46.7 ± 3.2 mm for 6 oviducts + 6 uteri) by the number of hours from ovulation to implantation (108 h). However, this average speed is very low and misses the higher punctuated speeds when velocity is above average. For example, in humans, 90% of the time spent in the oviduct is in the ampulla or at the ampulla-isthmus junction [19]. Higher rates of speed occur when the embryo is released and during back-and-forth movement. In some species, there is also an arrest in embryo movement at the utero-tubal junction, suggesting higher punctuated velocities when the embryo is moving. Indeed, punctuated velocities of maxima from 6.5 to 29.7 µm/sec have been observed in the oviduct for microspheres emulating the size of embryos (H. Croxatto, personal communication).

With such a potently lethal form of stress, why would SS not be lethal to the embryo moving in the oviduct or uterus? The higher density of the embryo compared with aqueous media yields an approximate 10–1 dynes/cm2 for the punctuated velocity maximum in the oviduct. The 261 ± 32.2-µm/sec velocities for embryos in the experimental SS are about 10-fold greater in magnitude than those measured in the oviduct, and presumably the 1.2-dynes/cm2 experimental SS is also 10-fold greater than for embryos in the oviduct. The duration of velocities resulting in death is 12 h in vitro, but the duration of maximal velocities of embryos in the oviduct is shorter. It is presumed that the SS acting on embryos in the oviduct are at low velocities or at high velocities for short periods that do not harm embryos in vivo.

In fact, some media movement over embryo surfaces has been considered a parameter in improving in vitro fertilization [56]. Fluid movement over an embryo in a microfluidics channel in vitro improved embryo development [57], but the velocity and SS were not measured. Presumably, the media velocity and shear created were similar to in vivo or simply created fluid flow rates that optimized gas and nutrient exchanges while not creating any negative effects from shear.

This 1.2-dynes/cm2 SS magnitude is a little lower than the shear measured for endothelial cells in arterioles that routinely sustain 10–44-dynes/cm2 shear stress [58]. It is likely that protection by the zona pellucida against SS would result in low levels of phosphorylated MAPK8/9 observed in embryos ex vivo [21, 23] but that conditions in the oviduct might create shear high enough to threaten an embryo with no zona pellucida.

MAPK8/9 Plays a Role in TUNEL and the Mechanism for Apoptosis in SS Embryos

It is unclear how SS is causal in the induction of MAPK8/9 and/or apoptosis. However, SS has been reported to induce MAPK8/9 in endothelial cells, which then induces growth arrest or apoptosis [5, 15, 59]. In one report, MAPK8/9 was induced by SS for a transient period, ending by 2 h [15] with an SS magnitude larger but similar to that in this study (12 vs. 1.2 dynes/cm2). However, a prolonged MAPK8/9 induction of 12 h with colchicine was required to induce apoptosis in endothelial cells. In the current study, the induction of MAPK8/9 phosphorylation was also prolonged, continuing throughout the time period leading up to 12 h, when TUNEL was elevated in about 40% of cells and the embryo was committed to death. This suggests that SS induces prolonged MAPK8/9 that contributes to TUNEL/apoptosis in the embryo, and the decrease in TUNEL by the MAPK8/9 inhibitor D-JNKI1 supports this conclusion.

SS Is More Potent in Inducing Lethality Than High Doses of Hyperosmolar Stress Induced by Sorbitol

The literature on hyperosmolar stress suggests that 500-1000 mM sorbitol causes rapid apoptosis within hours of induction in somatic cells [60–62] and that even 200 mM sorbitol can cause apoptosis within a day of induction in some cell lines. However, embryos are very resistant to even 1000 mM (1403 mOs) sorbitol, dying between 24 and 48 h. Shear stress, however, causes death by 12 h, 8 h if the zona pellucida is absent. This acceleration of lethality, from between 24 and 48 h to 12 h, suggests that SS is potently lethal.

A Novel Role of the Zona Pellucida

This study suggests additional reasons for the existence of the zona pellucida, which is thought primarily to prevent fertilization and lessen the chance of polyspermy. Many postfertilization functions for the zona pellucida have been proposed and corroborated with data, such as the general protection of the embryo from viruses and bacteria and the filtering of luminal molecules [63, 64]. This report adds a new reason for the zona pellucida to envelop the embryo for nearly 5 days after fertilization.

The zona pellucida may dampen SS during embryonic movement along the oviduct and uterus. In previous studies, it was shown that eight-cell-stage embryos that had their zona pellucida removed were healthy in culture or if cultured to the blastocyst stage and reimplanted into the uterus [65]. But if embryos with the zona pellucida removed were immediately reimplanted into the oviduct, no embryos survived. This could be because they were retained in the oviduct or because movement in the oviduct resulted in SS that was lethal. However, Bronson and McLaren took care to reduce the zona pellucida, leaving a small residual amount so that the embryos could be handled without stickiness. The current study concurs with the previous report on two points: 1) removing the zona pellucida becomes lethal if embryonic movement occurs after removal, and 2) the E2.5 embryo is more sensitive to zona pellucida removal than the E3.5 embryo.

Shear stress may mediate two important sublethal effects on the outside surface of the embryo. First, hyperosmolar stress reduces embryo growth rates through the MAPK8/9 function, and SS-activated MAPK8/9 may have similar effects since SS also reduces cell number. Dampening SS would increase growth rates. A second reason to dampen the MAPK8/9 response prior to implantation is that stress and stress enzymes may trigger postimplantation differentiation. For example, hyperosmolar stress activates chorionic somatomammotropin hormone 1 (Csh1, formerly known as placental lactogen [Pl]-1) and proliferin (Plf) mRNA in TS cells, essential postimplantation events in placental cells (Liu et al., unpublished data). Additionally, the transcription factor HAND1 and CSH1 protein are induced by hyperosmolar stress in a MAPK8/9-dependent manner (Zhong et al., unpublished data). Supporting this, an element of the induction of maternal recognition of pregnancy, rapid inhibitor of DNA binding 2 (ID2) nuclear export in response to hyperosmolar stress, is shared by differentiating TS cells and trophectodermal cells in preimplantation embryos. Therefore, the zona pellucida may sustain higher growth rates and prevent premature differentiation in preimplantation trophoblasts.

SS in Embryos and Endothelial Cells

Use of the RCCS1 microgravity simulator to create shear provided the advantage of an empirically measurable amount of shear without requiring adhesion that is not normal to preimplantation embryos. Such adhesion is normal for endothelial cells in experimental shear testing. The setup of the RCCS1 apparatus constrained the embryo shear testing to a single dose. That single dose was produced when motion, viscosity, and buoyancy forces balanced and created a stationary aqueous microdrop. Although shear dose dependence could not be measured, time dependence could be measured. The threshold of 12 h of MAPK8/9 phosphorylation required for induction of apoptosis is the same as for endothelial cells. This suggests that the molecular mechanisms in embryos and endothelial cells for inducing MAPK8/9 and apoptosis may be shared.

Finally, SS is a physical process unique to the outside cells of the embryos and could provide an auxiliary cue to the "outsidedness" that was proposed as a cause of lineage determination in the inside-outside hypothesis [66]. Shear stress is a unique physical force that may act to modulate several important developmental mechanisms in peri-implantation embryos.

ACKNOWLEDGMENTS

We are indebted to Dr. Josh Zimmerberg for loaning two additional RCCS-1s and numerous HARVs that greatly accelerated our work. We thank Mike Kruger for advice on statistical analysis. We thank PrimaProof for excellent editorial assistance. We are also indebted to Dr. Richard Tasca, Dr. Anne McLaren, and Dr. Horacio Croxatto for helpful discussions about the data and criticisms of the manuscript. We also thank Drs. Roger Pedersen, Ann Sutherland, and Richard Gardner for discussing unpublished data.

FOOTNOTES

1 Supported by grants from NASA (NRA-NAG2–1503) and the National Institute of Child Health and Human Development, NIH (R01-HD40972). Back

2 Correspondence: D.A. Rappolee, CS Mott Center for Human Growth, Wayne State University School of Medicine, 275 East Hancock, Detroit, MI 48201. FAX: 313 577 8554; drappole{at}med.wayne.edu Back

Received: 16 December 2005.

First decision: 16 January 2006.

Accepted: 28 March 2006.

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