Developmental Changes in Expression of Genes Involved in ...

BIOLOGY OF REPRODUCTION 84, 43–51 (2011) Published online before print 1 September 2010. DOI 10.1095/biolreprod.110.086249

Developmental Changes in Expression of Genes Involved in Regulation of Apoptosis in the Bovine Preimplantation Embryo1 Justin M. Fear and Peter J. Hansen2 Department of Animal Sciences and D. H. Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, Florida

The early bovine preimplantation embryo is resistant to proapoptotic signals until around the 8- to 16-cell stage. We hypothesized that 2-cell embryos have higher amounts of antiapoptotic proteins and lower amounts of proapoptotic proteins when compared to embryos 16 cells. Steady-state concentrations of mRNA for the antiapoptotic genes BCL2 and HSPA1A were higher for MII oocytes, 2-cell embryos, and 2-cell embryos treated with alpha-amanitin as compared to 16-cell embryos. Steady-state concentrations of mRNA for the proapoptotic gene BAD increased in embryos 16 cells. There was no significant effect of stage of development on steady-state mRNA concentrations of BCL2L1, DFFA, or BAX. Using immunohistochemistry, it was found that BCL2 was present in greater relative concentrations for 2-cell embryos than for embryos 16 cells. These results were confirmed by Western blotting. Relative amounts of immunoreactive BAX detected by immunofluorescence were lower for 2-cell embryos than for embryos 16 cells. Using Western blotting, a high molecular weight (46 kDa) form of BAX was highest in 16-cell embryos, intermediate in 2-cell embryos, and lowest in MII oocytes. There were no effects of stage of development on relative amounts of immunoreactive BCL2L1, HSPA1A, or BAD, as determined by immunofluorescence. Treatment of embryos with alpha-amanitin from Day 0 to Day 5 or Day 4 to Day 5 after insemination reduced activation of group II caspases and terminal deoxynucleotidyl transferase dUTP nick end labeling after treatment with the proapoptotic signal C2 ceramide at Day 5 after fertilization. Thus, transcription of BAX or other proteins is required for acquisition of the capacity for apoptosis. Results support the idea that changes in amounts of BCL2 family members are important for the inhibition of apoptosis in the 2cell embryo and in the establishment of the capacity for apoptosis later in development. apoptosis, BAD, BAX, BCL2, embryo, HSPAIA, preimplantation embryo

INTRODUCTION Exposure of preimplantation embryos to a variety of cellular stresses can induce apoptosis in all or a fraction of blastomeres. 1 Supported by National Research Initiative competitive grant no. 200735203-18073 from the USDA National Institute of Food and Agriculture. 2 Correspondence: Peter J. Hansen, PO Box 110910, Department of Animal Sciences, University of Florida, Gainesville, FL 32611-0910. FAX: 352 392 5595; e-mail: [email protected]

Received: 27 May 2010. First decision: 9 July 2010. Accepted: 26 August 2010. Ó 2011 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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Among the conditions that induce apoptosis in the bovine embryo are heat-shock [1, 2], arsenic [3], pro-oxidants [4–6], and tumor necrosis factor (TNF)-a [7, 8]. The consequences of apoptosis on the developmental competence of the embryo are dependent on the extent of its induction. Induction of apoptosis in up to 30% of blastomeres, as occurs after TNF administration [7], has no effect on development to the blastocyst stage. In fact, apoptosis may allow the embryo to survive stress by removal of damaged cells. Inhibition of apoptosis by the addition of the group II caspase inhibitor z-DEVD-fmk exacerbated the deleterious effect of heat shock on development to the blastocyst stage [2, 9]. Extensive apoptosis, as occurs after inhibition of survivin synthesis through RNA interference [10], leads to a block in development. Induction of apoptosis is developmentally regulated. In cultured bovine embryos, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells are first seen between the six- and eight-cell stage [11, 12]. Similarly, acquisition of an apoptotic response to heat shock [2] and TNF [7] also occurs after the eight-cell stage. Heat shock induces apoptosis through the mitochondrial or intrinsic pathway by activation of caspase 9 and group II caspases, such as caspase 3 [1–3, 8, 13]. The induction of apoptosis by heat shock, as shown by TUNEL, can be blocked by the addition of caspase 9 or group II caspase inhibitors [2, 8]. Interestingly, TNF also acts in the bovine preimplantation embryo through a caspase 9dependent pathway [8]. The inhibition of apoptosis in early cleavage stage embryos involves at least two blocks in the intrinsic pathway. The mitochondrion itself is resistant to depolarization, because neither heat shock [1, 13] nor ceramide [14] (a putative signaling molecule for activation of the intrinsic pathway) induce caspase 9 or group II caspase activation in the 2-cell embryo. In addition, the nucleus is resistant to caspasemediated TUNEL. Artificial activation of the intrinsic pathway by carbonyl cyanide 3-chlorphenylhydrazone (CCCP), a chemical inducer of mitochondrial depolarization, activated caspase 9 and group II caspases in 2-cell embryos, but did not result in DNA fragmentation [13]. Lack of TUNEL in response to mitochondrial depolarization is caused, at least in part, by epigenetic modifications that can be reversed by interfering with DNA methylation or histone deacetylation [15]. The objective of the present study was to determine the molecular basis for the developmental changes in mitochondrial and nuclear responses to proapoptotic signals. We hypothesized that the 2-cell embryo contains more antiapoptotic proteins and less proapoptotic proteins as compared to the 16-cell embryo. The focus was on several members of the BCL2 family of proteins regulating mitochondrial permeability in response to proapoptotic signals [16], heat shock protein 70 (HSPA1A), which can inhibit mitochondrial depolarization by interrupting the BCL2 family signaling cascade [17], and can inhibit caspase 3 activity [18], and DFFA (also called inhibitor

ABSTRACT

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of caspase-activated DNase), which is cleaved during apoptosis to activate caspase-activated DNase (DFFB) [19]. MATERIALS AND METHODS Materials for Embryo Culture

In Vitro Production of Embryos Embryo production was performed as previously described [21]. Briefly, a mixture of beef and dairy cattle ovaries were obtained from a local abattoir (Central Beef Packing Co., Center Hill, FL) and cumulus-oocyte complexes (COCs) were collected by slicing follicles that were 2- to 10-mm in diameter on the surface of ovaries. COCs containing at least one layer of compact cumulus cells were selected for subsequent steps. The COCs were washed twice in oocyte collection medium and placed in groups of 10 in 50-ll drops of oocyte maturation medium with a mineral oil overlay, and matured for 20–22 h at 38.58C and 5% CO2 in humidified air. Matured oocytes were then washed twice with Hepes-TALP and transferred in groups of 200 to a 35-mm 3 10-mm petri dish containing 1.7 ml IVF-TALP, 80 ll PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 lM epinephrine in 0.9% [wt/vol] NaCl), and 120 ll Percoll-purified spermatozoa (17 3 106 sperm/ml) from a pool of frozenthawed semen (three to four bulls; a separate pool of semen was used for each replicate) for a final concentration of approximately 1.2 3 106 sperm/ml at fertilization. After 8–10 h of coincubation at 38.58C and 5% CO2 in humidified air, putative zygotes were removed from fertilization plate and denuded of cumulus cells by vortexing in 100 ll hyaluronidase (1000 U/ml in HepesTALP). Thereafter, embryos were cultured in groups of 25 to 30 in 50-ll drops of medium overlaid with mineral oil at 38.58C, 5% CO2, 5% O2, ;90% N2, with humidified air. For immunocytochemistry and Western blotting experiments, embryos were cultured in KSOM-BE2. For real-time quantitative real-time RT-PCR (qPCR) experiments, embryos were cultured in KSOM-BE2 or KSOM-BE2 þ 50 lM a-amanitin. For the experiment to test the effect of aamanitin on group II caspase activity and TUNEL, embryos were cultured in KSOM-BE2 or KSOM-BE2 þ 50 lM a-amanitin, as described in Experimental Design.

RNA Extraction Oocytes were collected after 22 h of maturation, 2-cell embryos and 2-cell embryos treated with a-amanitin were collected between 28 and 29 h postinsemination (hpi), and 16-cell embryos were collected at Day 5 postinsemination. Total RNA was extracted using the PicoPure RNA isolation kit (Molecular Devices, Sunnyvale, CA) following the manufacturer’s instructions. Briefly, 100 ll of extraction buffer was added to 20 oocytes or embryos and the mixture incubated at 428C for 30 min. Thereafter, 100 ll of 70% (vol/vol) ethanol was added, and the mixture added to a preconditioned RNA purification column. After a series of three washes using two different wash buffers, RNA was eluted using 15 ll of elution buffer in a clean 1.5-ml microcentrifuge tube. RNA concentration was determined using a NanoDrop 2000 (Thermo Fisher Scientific Inc., Waltham, MA), and samples were stored at 808C until analysis.

Primers used for qPCR were manufactured by Integrated DNA Technologies (Coralville, IA), and are described in Table 1. To remove contaminating DNA, RNA samples were treated with DNase I (New England Biolabs, Ipswich, MA) according to manufacturer’s specifications. Briefly, 10 ll RNA sample was treated with 10 ll DNase mix (1 ll DNase I, 2 ll DNase buffer, and 7 ll 0.1% [vol/vol] diethyl pyrocarbonate treated H2O [DEPC]) at 378C for 30 min, followed by 758C for 15 min. Complimentary DNA was produced using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA), according to the manufacturer’s directions. DNase-treated samples (20 ll) were diluted to a final volume of 35 ll with DEPC H2O. Reverse transcription was performed in 15-ll reactions by adding 7.5 ll diluted DNase-treated sample to either 7.5 ll of RT( þ) master mix (3.15 ll DEPC H2O, 1.5 ll 103 reaction buffer, 1.5 ll 103 random primers, 0.6 ll 253 dNTPs, and 0.75 ll 203 RTase) or 7.5 ll of RT() master mix (3.9 ll DEPC H2O, 1.5 ll 103 reaction buffer, 1.5 ll 103 random primers, and 0.6 ll 253 dNTPs). Reaction conditions were 258C for 10 min, 378C for 2 h, and 858C for 5 min. Complimentary DNA from a control cell line (bovine BEND cells) was used to produce a standard curve to test efficiency of each primer. After reverse transcription, samples were diluted using fivefold dilutions to create a 5-point standard curve. Standard curves were performed in a 25-ll reaction volume (12.5 ll 23 SYBR Geen PCR master mix [Applied Biosystems], 0.75 ll each of 10 mM forward and reverse primers, and 8.5 ll DEPC H2O) using an ABI 7300 instrument (Applied Biosystems). The conditions for amplification were 1 cycle for 10 min at 958C, followed by 50 cycles of 15 sec at 958C and 1 min at 608C. Cycle threshold (Ct) values were plotted against the log10 of the template concentration and primers used if the slope was between 2.9 and 3.7, with an R2 .0.95. In addition, agarose gel electrophoresis was performed to verify synthesis of a single product at the appropriate size. Complimentary DNA from multiple RT( þ) reactions, from the same pool of 20 oocytes or embryos, were pooled together to ensure adequate cDNA. Each real-time plate consisted of one replicate of all genes in all sample groups (oocyte, 2-cell, 2-cell þ a-amanitin, and 16-cell embryos). Samples were analyzed in duplicate in 25-ll reactions (12.5 ll 23 SYBR Geen PCR master mix, 0.75 ll each 10 mM forward and reverse primers, and 8.5 ll DEPC H2O) using an ABI 7300 PCR machine (Applied Biosystems). Amplification proceeded for 1 cycle for 10 min at 958C, followed by 50 cycles of 15 sec at 958C and 1 min at 608C.

Immunocytochemistry Two-cell embryos were collected between 28 and 29 hpi, and 16-cell embryos were collected at Day 5 postinsemination. Two-cell embryos and 16-cell embryos were washed four times in 50-ll drops of 10 mM KPO4 (pH 7.4) containing 0.9% (wt/vol) NaCl (PBS) and 1 mg/ml polyvinylpyrrolidone (PBS-PVP) by transferring from drop to drop. Embryos were fixed for 15 min in PBS containing 4% (vol/vol) paraformaldehyde. After fixation, embryos were washed in PBS-PVP and placed in 500 ll of PBS-PVP and stored at 48C for up to 1 week. Antibodies were affinity-purified rabbit polyclonal antibodies against BCL2 (Santa Cruz Biotechnology, Santa Cruz, CA), BCL2L1 (AbCam Cambridge, MA), BAX (Santa Cruz Biotechnology), and BAD (Assay Designs, Ann Arbor, MI), or a purified mouse monoclonal antibody for HSPA1A (Chemicon International). Either rabbit IgG or mouse IgG (Sigma-Aldrich) was used as a negative control. Each antibody was used at a concentration determined to be optimal in preliminary experiments for measuring fluorescence intensity differences (BCL2, 4 lg/ml; BCL2L1, 10 lg/ml; HSPA1A, 5 lg/ml; BAX, 2 lg/ml; BAD, 10 lg/ml). Antibodies were labeled using the Zenon 488 labeling kit (Invitrogen), following the manufacturer’s instructions. For every 1 lg antibody or nonspecific IgG being labeled, 5 ll of the Zenon Fab fragment mixture was added and incubated for 5 min in the dark. An equal mass of Zenon nonspecific IgG was then added to block unbound Fab fragments and incubated for 5 min in the dark. Two-cell and 16-cell embryos were permeabilized in groups of 30–50 in 500 ll PBS containing 0.2% (vol/vol) Triton-X (Sigma-Aldrich) for 30 min at room temperature. Embryos were washed four times in PBS-PVP and placed into 50 ll PBS containing 20% (vol/vol) normal goat serum (Pel-Freez Biologicals, Rogers, AR) for at least 1 h at room temperature. After blocking, embryos were briefly washed in 1 drop of PBS-PVP and then transferred to a drop of Zenon-labeled antibody or labeled IgG, and incubated for 1 h at room temperature. Embryos were then washed four times, fixed for 15 min in 4% (vol/vol) paraformaldehyde, and nuclei labeled with Hoescht 33342 (1 lg/ml; Sigma-Aldrich) for 15 min. Embryos were washed four to five times, mounted on a microscope slide using ProLong Gold Anti-Fade mounting medium (Thermo Fisher Scientific Inc), and fluorescence visualized using a Zeiss


Media for in vitro production of embryos were obtained as follows. HepesTyrodes lactate (TL) and IVF-TL were purchased from Millipore (Billerica, MA) or Caisson Laboratories, Inc. (North Logan, UT) and used to prepare Hepes-Tyrodes albumin lactate pyruvate (TALP) and IVF-TALP, as described by Parrish et al. [20]. Oocyte collection medium was Tissue Culture Medium 199 (TCM-199) with Hanks salts without phenol red (Atlanta Biologicals, Lawrenceville, GA) supplemented with 2% (vol/vol) bovine steer serum (PelFreez Biologicals, Rogers, AR) containing 2 U/ml heparin, 100 U/ml penicillinG, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocyte maturation medium was TCM-199 (Gibco, Invitrogen, Carlsbad, CA) with Earles salts supplemented with 10% (vol/vol) bovine steer serum, 2 lg/ml estradiol 17-b, 20 lg/ ml bovine FSH (Folltropin-V; Agtech Inc., Manhattan, KS), 22 lg/ml sodium pyruvate, 50 lg/ml gentamicin sulfate (Sigma-Aldrich, St. Louis, MO), and 1 mM L-glutamine or alanyl-glutamine. Percoll was from GE Healthcare (Uppsala, Sweden). Frozen semen from various bulls was donated by Southeastern Semen Services (Wellborn, FL). Potassium simplex optimized medium (KSOM), containing 1 mg/ml bovine serum albumin (BSA), was obtained from Millipore or Caisson Laboratories Inc. KSOM was modified with 3 mg/ml essentially fatty-acid free BSA (Sigma-Aldrich), 2.5 mg/ml gentamicin, 100 U/ml penicillin-G, and basal medium Eagle nonessential amino acids (Sigma-Aldrich), to produce KSOM-bovine embryo 2 (BE2), as described elsewhere [21].

Quantitative Real-Time PCR

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DEVELOPMENTAL REGULATION OF APOPTOSIS TABLE 1. Primer sets used for qRT-PCR. Gene

Accession no.

BCL2

XM_586976.4

BCL2L1

NM_001077486.2

HSPA1A

NM_174550.1

DFFA

NM_001075342.1

BAX

NM_173894.1

BAD

NM_001035459.1

DFFB

NM_001035109.1

HIST1H2A

U62674

Sequence

Tm (8C)

Product size (bp)

5 0 -TCGTGGCCTTCTTTGAGTTC-3 0 5 0 -CGGTTCAGGTACTCGGTCAT-3 0 5 0 -CTCAGAGTAACCGGGAGCTG-3 0 5 0 -CTGGGGGTTTCCATATCTGA-3 0 5 0 -GGGGAGGACTTCGACAACAGG-3 0 5 0 -CGGAACAGGTCGGAGCACAGC-3 0 5 0 -GGTGCTTGACCAGAGAGAGG-3 0 5 0 -CCAAGGTCAGCTCTGGACTC-3 0 5 0 -CTCCCCGAGAGGTCTTTTTC-3 0 5 0 -TCGAAGGAAGTCCAATGTCC-3 0 5 0 -CTTTTCTGCAGGCCTTATGC-3 0 5 0 -GGTAAGGGCGGAAAAACTTC-3 0 5 0 -CCCTGCATAGCGAGAAGAAG-3 0 5 0 -ATTCCGTGCCATCCTCATAG-3 0 5 0 -GTCGTGGCAAGCAAGGAG-3 0 5 0 -GATCTCGGCCGTTAGGTACTC-3 0

60

109

60

142

60

245

60

120

60

176

59

151

60

129

57

182

Western Blotting Oocytes were collected after 22 h of maturation, 2-cell embryos were collected between 28 and 29 hpi, and 16-cell embryos were collected at Day 5 postinsemination. Before collecting oocytes, COCs were washed twice with Hepes-TALP and then denuded of cumulus cells by vortexing in 100 ll hyaluronidase (1000 U/ml in Hepes-TALP). Denuded oocytes and embryos were washed four times in 50-ll drops of PBS-PVP by transferring from drop to drop. Groups of 25–100 ooctyes/embryos were placed in a prewarmed 50-ll drop of 0.1% (wt/vol) protease from Streptomyces griseus in PBS for 2–5 min on a slide warmer to digest the zona pellucida. Groups of 200 oocytes or embryos were then placed into 1.5-ml microcentrifuge tubes containing 1 ml PBS-PVP. Samples were centrifuged at 13 600 3 g, supernatant removed, and samples stored at 808C. Oocyte and embryo samples (200) were placed in 20 ll loading buffer (0.125 M Tris-HCl [pH 6.8] containing 20% [vol/vol] sucrose, 10% [wt/vol] SDS, trace amounts of bromophenol blue, and 5% [vol/vol] 2-mercaptoethanol). Samples were heated at 958C for 5 min and cooled on ice for 1 min before performing one-dimensional, discontinous SDS-PAGE using a Ready Gel 4– 15% (wt/vol) Tris-HCl (Bio-Rad, Hercules, CA) gel. Conditions for electrophoresis were 125 V/40 mA for 1–1.5 h at room temperature. Proteins were then transferred electrophoretically to a Hybond ECL 0.2-lm nitrocellulose membrane (GE Healthcare). Conditions for transfer were 30 V/90 mA for 12 h at 48C using a degassed mixture of 25 mM Tris, 193 mM glycine, and 20% (vol/vol) methanol. Membranes were blocked overnight in 10 mM Tris (pH 7.6), 0.87% (wt/vol) NaCl, and 0.3% (vol/vol) Tween-20 (TBS-T), that also contained 5% (wt/vol) nonfat dry milk (TBS-TM). Membranes were rinsed twice with TBS-T and incubated for 2 h at 48C with either a polyclonal antibody (BCL2 or BAX; Santa Cruz Biotechnology) or rabbit IgG at 1 lg/ml in TBS-TM. Membranes were washed four times with TBS-T, incubated for 1.5 h at 48C with horseradish peroxidase conjugated to goat ant-rabbit IgG (0.04 lg/ml; Santa Cruz Biotechnology) in TBS-TM, and then washed as above. Blots were exposed to the ECL Plus Western blotting chemilumines-

[43]

[22]

cence substrate kit (GE Healthcare) for 5 min and then exposed to film for 3–15 min. To allow reprobing, membranes were stripped using a stripping buffer (62.5 mM Tris-HCl, 2% [wt/vol] SDS, 100 mM 2-mercaptoethanol) for 30 min at 508C. Membranes were washed and reprobed as described before. Peptide neutralization was performed to verify the specificity of the two antibodies. Briefly, 5 lg/ml of the peptide used to produce antibody against BCL2 or BAX (Santa Cruz Biotechnology) was preincubated with 1 lg/ml of the respective antibody for 1 h, and the antibody mixture was used to probe blots as described above.

Assessment of Group II Caspase Activity and TUNEL Embryos were washed three times in 20 ll drops of prewarmed HepesTALP and then placed in 20 ll drops of prewarmed Hepes-TALP containing 5 lM PhiPhiLux-G1D2 (a group II caspase substrate specific for caspase 2, 3, and 7; OncoImmunin Inc., Gaithersburg, MD) at 38.58C for 1 h in a humidified box. Negative controls were incubated in Hepes-TALP. Embryos were then washed four times in Hepes-TALP and placed onto 2-well slides containing 100 ll Hepes-TALP. Images were then acquired using the Zeiss Axioplan microscope with a 103 objective and the FITC and DIC filter sets. Digital images were acquired using the AxioVision software and a high-resolution black and white AxioCam MRm digital camera (Zeiss). Images were acquired using the same exposure time within replicate, and focal plane was selected using DIC channel. Subsequently, images were scored for fluorescent intensity using an arbitrary scale of 0–5. After images for caspase activity were acquired, embryos were fixed for 15 min at room temperature in PBS containing 4% (vol/vol) paraformaldehyde, washed in PBS-PVP, placed in 500 ll of PBS-PVP, and stored at 48C for up to 1 week until analysis for TUNEL. For TUNEL, embryos were permeabilized in 500 ll PBS containing 0.5% (vol/vol) Triton-X for 1 h at room temperature. Embryos were washed four times in PBS-PVP, incubated in 20 ll RQ1 RNasefree DNase (50 U/ml) at 378C for 1 h in a humidified box, and then incubated in 20 ll of TUNEL reaction mixture (TMR red-conjugated dUTP and the enzyme terminal deoxynucleotidyl transferase; Roche Diagnostics Corporation) at 378C for 1 h in a humidified box. Negative controls were incubated in the absence of terminal deoxynucleotidyl transferase. Embryos were washed four times with PBS-PVP and incubated in 20 ll drop of Hoechst 33342 (1 lg/ml) for 15 min at room temperature. Embryos were washed four times in PBS-PVP and placed onto 2-well slides containing 100 ll PBS-PVP. Images were then acquired using the Zeiss Axioplan microscope (Zeiss) with a 103 objective and the rhodamine, DAPI, and DIC filter sets. Digital images were acquired using the AxioVision software and a high-resolution black and white AxioCam MRm digital camera (Zeiss). Images were acquired using the same exposure time within replicate, and focal plane was selected using DAPI channel. The number of nuclei (labeled with Hoechst 33342), and the number of TUNEL-positive nuclei (TMR red labeled) were counted for each individual embryo.

Experimental Design For all experiments, control drops were set aside to assess cleavage at Day 3 postinsemination and development to the blastocyst stage at Day 8 postinsemination. Only replicates with characteristic cleavage and blastocyst development rates were used for molecular or immunochemical analysis.


Axioplan microscope (Zeiss, Go¨ttingen, Germany) with a 403 objective, and the fluorescein isothiocyanate (FITC), 4 0 ,6 0 -diamidino-2-phenylindole (DAPI), and differential interference contrast (DIC) filter sets. Digital images were acquired using the AxioVision software and a high-resolution black and white AxioCam MRm digital camera (Zeiss). Images were acquired using the same exposure time within replicate, and focal plane was selected using the DIC channel. Analysis of immunocytochemical images was performed using ImageJ (National Institutes of Health, Bethesda, MA). Images of individual embryos were outlined by hand with the polygon selection tool. After selection, additional exclusion was made for areas that did not correspond to the embryo, as well as areas within the embryo between cells and, in cases where it occurred, in regions of the embryo that had broken through the zona pellucida. In the latter case, extrusion through the zona pellucida was a fixation artifact, and extruded areas were consistently associated with lower fluorescent intensity. Mean gray pixel intensity of the selected area was measured using the FITC channel. Background and nonspecific binding was removed by averaging mean pixel for IgG controls, and subtracting these values from individual mean pixel intensities of embryos in corresponding stages. Adjusted mean pixel intensities that were below zero were set to zero.

Reference

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FIG. 1. Quantitative real-time RT-PCR for antiapoptotic genes BCL2 (A), BCL2L1 (B), HSPA1A (C), and DFFA (D), proapoptotic genes BAX (E) and BAD (F), and the housekeeping gene HIST1H2A (G). Data on steady-state mRNA concentrations for bovine MII oocytes, 2-cell embryos, 2-cell embryos treated with a-amanitin, and 16cell embryos are expressed as fold change relative to MII oocytes (least-squares means 6 SEM). Reactions were performed with cDNA samples (n ¼ 5) from individual groups of 20 oocytes or embryos. Bars with different letters differ (P , 0.05).


Quantitative PCR. Treatments included MII oocytes, 2-cell embryos, 2-cell embryos treated with 50 lM a-amanitin, and 16-cell embryos. Oocytes and embryos were collected and analyzed in groups of 20. Eight genes were analyzed for each treatment: 4 antiapoptotic genes (BCL2, BCL2L1, HSPA1A, and DFFA), 3 proapoptotic genes (DFFB, BAX, and BAD), and HIST1H2A as a housekeeping gene [22]. The qPCR experiments were replicated with 5 separate pools of embryos for each stage. Immunocytochemistry. For each antibody, treatments included 2-cell embryos and 16-cell embryos. Each replicate consisted of at least 10 2-cell and 10 16-cell embryos for IgG-negative controls, and at least 10 2-cell and 10 16-cell embryos for antibody labeling (BCL2, BCL2L1, HSPA1A, BAX, BAD). Experiments were replicated four to eight times. The total number of embryos used was as follows: BCL2, n ¼ 111; BCL2L1, n ¼ 183; HSPA1A, n ¼ 239; BAX, n ¼ 142; BAD, n ¼ 186. Western blotting. A total of 4 Western blots were performed. Treatments included on all blots were MII oocytes, 2-cell embryos, and 16-cell embryos. Membranes were stripped to allow for blotting with different antibodies. Effect of a-amanitin on ceramide-induced group II caspase assay and TUNEL. Embryos received 1 of 3 treatments. One group was cultured with KSOM-BE2 from Day 0 to Day 5.5, the second was cultured in KSOM-BE2 containing 50 lM a-amanitin from Day 0 to Day 5.5, and the third was cultured in KSOM-BE2 from Day 0 to Day 4 and KSOM-BE2 containing 50 lM aamanitin from Day 4 to Day 5.5. On Day 4, all embryos were moved to fresh microdrops containing 50 ll of fresh KSOM-BE2 6 a-amanitin. At Day 4.5, embryos were moved to 400 ll of KSOM-BE2 that contained 50 lM C2

ceramide (added to induce apoptosis [14]) and the respective a-amanitin treatment. Embryos were cultured in 5-well plates (Minitube, Verona, WI) without a mineral oil overlay. Caspase and TUNEL were measured after 24 h of culture on Day 5.5. The experiment was replicated four times with a total of 300 embryos.

Statistical Analysis Data on mRNA abundance were subjected to least-squares analysis of variance using the general linear models procedure (GLM) of the Statistical Analysis System (SAS for Linux, Release 9.2; SAS Institute Inc., Cary, NC). PCR data were analyzed using DCt. In preliminary analysis using Ct values, HIST1H2A had significant variation between treatments. Accordingly, DFFB was used instead as a housekeeping gene, because it was expressed similarly between treatments. The p-diff procedure with Tukey adjustment was used as a means separation test. The DDCt value for each gene was calculated by the difference between the DCt of the embryo groups and the DCt of MII oocytes. Data are reported as fold change calculated by 2DDCt. Data on adjusted pixel intensity from immunocytochemical analysis were also analyzed by least-squares analysis of variance using PROC GLM. Replicate was considered as a fixed effect, and the p-diff procedure was used as a means separation test. Data on effects of a-amanitin on cell number, group II caspase activity, and percentage of nuclei positive for TUNEL were analyzed by least-squares

DEVELOPMENTAL REGULATION OF APOPTOSIS

47

analysis of variance using PROC GLM. Replicate was considered as a random effect, and the p-diff procedure was used as a means separation test.

RESULTS Quantitative Real-Time RT-PCR A total of 8 genes were analyzed by qPCR, including antiapoptotic genes (BCL2, BCL2L1, HSPA1A, and DFFA), proapoptotic genes (BAX, BAD, and DFFB), and HIST1H2A. DFFB did not vary between stages, and was used as a housekeeping gene for calculation of DCt. Differences in steady-state concentrations of mRNA for each gene are shown in Figure 1. Of the antiapoptotic genes, BCL2 steady-state mRNA did not differ significantly different between MII oocytes, 2-cell embryos, and a-amanitin-treated 2-cell embryos, but was lower in 16-cell embryos compared with the earlier stages (Fig. 1A; P , 0.0001). There was no significant effect of stage of development on steady-state mRNA concentrations of BCL2L1 (Fig. 1B). Patterns of mRNA concentrations for HSPA1A (Fig. 1C) and DFFA (Fig. 1D) were similar to those for BCL2, with a reduction in concentration in embryos 16 cells. This difference was

significant for HSPA1A (P ¼ 0.0005), but not for DFFA. For the proapoptotic genes, steady-state concentrations of BAX were not affected by stage (Fig. 1E). However, steady-state concentrations of BAD had a distinct temporal pattern, with a significant increase in mRNA levels in embryos 16 cells (Fig. 1F; P , 0.0001). Immunocytochemistry and Western Blotting Results for BCL2 are shown in Figure 2. Relative fluorescent intensity of BCL2 determined by immunofluorescence was greater for 2-cell embryos than for embryos 16 cells (compare Fig. 2B with Fig. 2E). Adjusted mean pixel intensity of immunoflourescence was greater (P ¼ 0.001) for 2cell embryos (Fig. 2G). These results were confirmed by Western blotting in three separate replicates. Intensity of the 26-kDa BCL2 immunoreactive band was more intense for 2cell embryos than for oocytes or 16-cell embryos (Fig. 2H). Immunolabeling of BCL2 was eliminated when antibody was coincubated with a BCL2 peptide (Supplemental Fig. S1 available at www.biolreprod.org).


FIG. 2. Immunoreactive amounts of BCL2 in 2-cell and 16-cell in vitro-produced embryos as determined by immunocytochemistry (A–G) and confirmed by Western blotting (H). Immunocytochemical results are representative images of 2-cell embryos (A–C) and 16-cell embryos (D–F). Shown are merged fluorescent images of Zenon 488-labeled anti-BCL2 (green) and nuclei labeled with Hoescht 33342 (blue) (B, C, E, F), or merged images of fluorescent labeling with differential interference contrast (A and D). Images of labeling with the negative control (nonspecific IgG labeled with Zenon 488) are in C and F. Bar ¼ 100 lm. The average pixel intensity of fluorescence associated with labeling with anti-BCL2, after correction for nonspecific labeling, is shown in G (least-squares means 6 SEM). Intensity of fluorescence was higher for 2-cell embryos, as indicated by different letters (P ¼ 0.001). Results of Western blotting for BCL2 in MII oocytes (lane 1), 2-cell embryos (lane 2), and 16-cell embryos (lane 3) are shown in H. The arrow indicates the band of interest at 26 kDa. All lanes of oocytes and embryos represent 200 oocytes or embryos.

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Shown in Figure 3 are immunoreactive amounts of BAX using immunocytochemistry (Fig. 3, A–G) and Western blotting (Fig. 3H). The difference in relative fluorescent intensity of BAX was lower for 2-cell embryos than for embryos 16 cells, as seen in representative images (compare Fig. 3B with Fig. 3E) and in analysis of pixel intensity of immunofluorescence (Fig. 3G) where pixel intensity was lower (P ¼ 0.0001) for 2-cell embryos (Fig. 3H). By Western blotting, three immunoreactive bands were detected, all of which could be eliminated or greatly reduced by coincubation of antibody with BAX peptide (Supplemental Fig. S1 available at www.biolreprod.org). One band was of expected size (23 kDa), another was a larger band of 46 kDa that presumably represents BAX dimmers, and a third, low-molecular weight band present in some samples was probably a proteolytic cleavage product. Differences between stages in amounts of the 23-kDa BAX were variable. However, the 46-kDa form was consistently present in highest amounts in 16-cell embryos, intermediate amounts in 2-cell embryos, and lowest amounts in MII oocytes.

There were no effects of stage of development on amounts of immunoreactive BCL2L1, HSPA1A, or BAD, as determined by immunofluorescence (Fig. 4). Reduction in Apoptosis in Ceramide-Treated Embryos Caused by a-Amanitin Treatment of embryos .16 cells at Day 5 after insemination with C2 ceramide causes induction of apoptosis [14]. To test whether capacity for apoptosis is dependent upon transcription prior to and on Day 5.5 after insemination, group II caspase activity and the percentage of cells labeling with the TUNEL reaction were determined for C2 ceramide-treated embryos in which transcription was blocked with a-amanitin from Day 0 to Day 5.5 or Day 4 to Day 5.5 after insemination. As expected, embryos cultured with a-amanitin were not able to develop past the 16-cell stage (i.e., the time of genomic activation in the bovine), so that cell number was lower (P , 0.01) in embryos cultured with a-amanitin from Day 0 to 5.5 or Day 4 to Day 5.5 than embryos cultured without a-amanitin (Fig. 5A). More-


FIG. 3. Immunoreactive amounts of BAX in 2-cell and 16-cell in vitro-produced embryos as determined by immunocytochemistry (A–G) and Western blotting (H). Immunocytochemical results are representative images of 2-cell embryos (A–C) and 16-cell embryos (D–F). Shown are merged fluorescent images of Zenon 488-labeled anti-BAX (green) and nuclei labeled with Hoescht 33342 (blue) (B, C, E, and F), or merged images of fluorescent labeling with differential interference contrast (A and D). Images of labeling with the negative control (nonspecific IgG labeled with Zenon 488) are in C and F. Bar ¼ 100 lm. The average pixel intensity of fluorescence associated with labeling with anti-BAX, after correction for nonspecific labeling, is shown in G (least-squares means 6 SEM). Intensity of fluorescence was lower for 2-cell embryos, as indicated by different letters (P , 0.0001). Results of Western blotting for BAX in MII oocytes (lane 1), 2-cell embryos (lane 2), and 16-cell embryos (lane 3) are shown in H. The arrows indicate the bands of interest at 46 and 23 kDa. All lanes represent 200 oocytes or embryos.


49 FIG. 4. Relative mean fluorescence intensity of BCL2L1 (A), HSPA1A (B), and BAD (C) in 2-cell and 16-cell in vitro-produced embryos. Data are the average pixel intensity of fluorescence associated with labeling with antibody, after correction for nonspecific labeling (least-squares means 6 SEM). There were no significant differences between 2-cell and 16-cell embryos.

DISCUSSION The ability of the preimplantation bovine embryo to undergo an apoptotic response is developmentally regulated with stressinduced apoptosis being blocked prior to the 8- to 16-cell stage [1, 3, 7, 13, 15]. The timing of acquisition of apoptosis responses is coincident with embryonic genome activation [1, 23], and we show here that capacity for apoptosis in embryos at Day 5 after insemination depends upon transcription, because treatment with a-amanitin reduced caspase activity and the percent of blastomeres that were TUNEL positive. The block to apoptosis is not caused by a lack of apoptotic machinery, because chemical depolarization of the mitochondria using CCCP leads to the activation of both caspase 9 and group II caspases [13]. Data from previous studies [13] indicate that the mitochondrion is a key organelle controlling developmental changes in apoptosis responses. At the 2-cell stage, mitochondria are resistant to depolarization caused by heat shock [1, 13] and ceramide [14], even though artificial depolarization of mitochondria with CCCP activated caspase 9 and group II caspases in 2-cell embryos. The present results support the idea that changes in amounts of BCL2 family members are important for resistance of the mitochondria to depolarization in the 2-cell embryo and in the establishment of the capacity for apoptosis later in development. Life and death of a cell is dictated by a balance of anti- and proapoptotic BCL2 family proteins [24]. Antiapoptotic BCL2 family members (BCL2 and BCL2L1) inhibit apoptosis by heterodimerizing with proapoptotic BCL2 family members (BAX, BAK, BOK). Heterodimerization prevents the proaptotic BCL2 family proteins from forming homodimers, which are required for mitochondrial pore formation and depolariza-

tion. Present results indicate that there is a switch from an abundance of BCL2 in early cleavage-stage embryos, which are not capable of apoptosis, to increased amounts of BAX in later-stage embryos, which possess the capacity for apoptosis. Quantitative real-time RT-PCR indicated higher amounts of BCL2 mRNA in oocytes and 2-cell embryos compared with 16-cell embryos. BCL2 also decreases from the zygote to the 2-cell embryo in the mouse [25], a temporal shift coincident with mouse genome activation [26]. In the human preimplantation embryo, mRNA for BCL2 is present in high amounts in the 2-cell embryo, and expression decreases in the 4- to 6-cell embryo coincident with genome activation [27]. Changes in immunoreactive amounts of BCL2 protein paralleled that for mRNA, with amounts higher in the 2-cell embryo than in the 16-cell embryo. This increase in BCL2 was not the result of transcription, because treatment with a-amanitin, a transcription inhibitor, had no effect on steady-state amounts of BCL2 mRNA. Therefore, it is most likely that amounts of BCL2 increase in the early cleavage embryo as the result of changes in translation of maternally derived mRNA. The high concentration of BCL2 in 2-cell embryos is likely to contribute to making the mitochondria refractory to stimuli leading to apoptosis. Further evidence for this idea is the fact that the maturing oocyte, which has lower amounts of BCL2, is capable of undergoing apoptosis in response to heat shock [28]. Steady-state mRNA of HSPA1A was also higher in the oocyte and 2-cell embryo than the 16-cell embryo. Immunoreactive concentration of HSPA1A protein remained constant through the 16-cell stage. Perhaps translation of HSPA1A is inhibited at early cleavage states by RNA-binding proteins. The bovine embryo has a variety of RNA-binding proteins, including staufen 1 and 2 and ELAVL1 [29]. ELAVL1 has been shown to bind to HSPA1A mRNA in the brain, and to regulate HSPA1A expression posttranscriptionally [30]. BCL2L1, another antiapoptotic member of the BCL2 family, did not show signs of developmental regulation in either mRNA or protein expression. The observation that


over, group II caspase activity (Fig. 5B) and the percent of blastomeres labeled with the TUNEL reagent (a measure of DNA fragmentation; Fig. 5C) were lower (P , 0.01) for embryos cultured with a-amanitin from Day 0 to Day 5.5 or Day 4 to Day 5.5.

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amounts of BCL2L1 mRNA and BCL2L1 protein are constant from the 2-cell stage through Day 5 of development confirms previous results in the bovine [31] and mouse [25]. Acquisition of capacity for apoptosis at the 16-cell stage is accompanied not only by a decrease in amounts of BCL2, but also by changes in proapoptotic BCL2 family members. There are two major classes of the proapoptotic BCL2 family members: multidomain proteins (BAX, BAK, BOK) and BH3-only domain proteins (BAD, BID, BIK, etc.). The multidomain members form heterodimers with the antiapoptotic proteins in the cytosol and mitochondrial membrane. Upon apoptotic stimuli, BH3-only members are activated, and


FIG. 5. Reduction in apoptosis in ceramide-treated embryos caused by a-amanitin. Embryos were cultured without a-amanitin or with a-amanitin from Day 0 to Day 5.5 or Day 4 to Day 5.5 after insemination. At Day 4.5 after insemination, embryos were transferred to medium containing C2 ceramide to induce apoptosis. Data are least-squares means 6 SEM of results for total cell number (A), group II caspase activity (B), and percent of nuclei that were TUNEL positive (C). Bars with different superscripts differ (P , 0.01).

either heterodimerize with antiapoptotic proteins (BCL2, BCL2L1, MCL1) to inhibit their function, or bind to the multidomain proapoptotic proteins to cause mitochondrial localization and pore formation [32]. In the current study, we investigated the multidomain member BAX and the BH3-only member BAD. Results indicated complex developmental changes for BAX protein and BAD mRNA. Steady-state mRNA for BAX did not vary between developmental stages. Similar results have been described in the mouse [25] and human [33]. In contrast, immunocytochemical analysis indicates not only an increase in immunoreactive amounts BAX at the 16-cell stage, but an increased amount of high-molecular weight BAX, as detected by Western blotting. Such results suggest posttranslational regulation of BAX that increases its synthesis and capacity for dimerization. One might have expected that dimeric proteins would disassemble under the denaturing and reducing conditions of SDS-PAGE. However, BAX and BAK1 belong to a class of amphipathic proteins called a-pore-forming proteins that contain several a helices involved in pore formation [34]. The a helical transmembrane domains of these proteins, and the extreme hydrophobic pockets that reside within these domains, allow pore-forming proteins to produce stable multimers even under reducing conditions [35]. BAD had the most interesting pattern of mRNA expression and protein concentration. The steady-state mRNA for BAD was very low at the 2-cell stage, and then increased almost 10 fold at the 16-cell stage. This same pattern is seen in the human preimplantation embryo, with very low levels of BAD mRNA until compaction, when there is an increase in expression [27, 33]. Despite the increase of BAD mRNA at the 16-cell stage, there was no change in immunoreactive amounts of BAD, as determined by immunocytochemistry. One possibility is that the 16-cell embryo is poised to synthesize additional amounts of BAD upon an apoptotic stimulus through translation of previously accumulated mRNA. BH3-only proteins have been shown to be regulated transcriptionally and posttranslationally. For example, BBC3 (previously PUMA) is transcriptionally upregulated by p53 [36] and forkhead box O3 [37] upon DNA damage or growth factor deprivation. Posttranscriptional regulation of BAD includes phosphorylation events at Ser112 [38, 39] and Ser136 [40], leading to BAD being sequestered by YWHAQ proteins [38, 40]. Upon apoptotic stimuli, these serines become dephosphorylated, and BAD is released from YWHAQ proteins and contributes to cell death [38]. One candidate for posttranscriptional regulation of mRNA for BAD is the newly described kinase, MAP4K3, that plays a role in DNA damage-induced cell death [41]. MAP4K3 functions by activating the mammalian target of rapamycin (mTOR) pathway. mTOR modulates the activity of eukaryotic translation initiation factors (EIF4B, EIF4E, EIF4F), which are involved in CAP-binding and mRNA stability [42]. It has been suggested that MAP4K3 stimulation of mTOR leads to the stabilization of BBC3 and BAD, and may contribute to enhancement of BBC3 and BAD translation. Apoptosis is a tightly regulated pathway, with multiple checks and balances to prevent accidental activation of cellular death without an appropriate signal. While caspases and DNases are the cellular executioners, it is the mitochondria, in concordance with the BCL2 family of proteins, that serve as judge and jury. The data presented here show that developmental regulation of mRNA and immunoreactive amounts of protein—in particular, BCL2, BAX, and BAD—are key factors in preventing mitochondrial depolarization and apoptosis in the


early preimplantation embryo, and for establishing the capacity for apoptosis at the 16-cell stage. ACKNOWLEDGMENTS The authors thank Dr. Alan Ealy for assistance with real-time qRT-PCR, William Rembert for obtaining ovaries, Marshall, Adam, and Alex Chernin and employees of Central Beef Packing Co. (Center Hill, FL) for donation of ovaries, and Scott A. Randell of Southeastern Semen Services (Wellborn, FL) for donating semen.

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