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a Department of Biology, Northeastern University, Boston, Massachusetts 02115
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The phenomenon of apoptosis has been studied in much greater detail in other cell types, such as lymphocytes and neurons. From these studies, basic mechanisms have emerged. Although the array of potential apoptotic signals is vast and varies with cell type, all apoptotic pathways appear to terminate in the activation of the caspase family of proteases [18–21]. Caspases are synthesized as inactive precursors that must be cleaved autocatalytically or by other caspases for activation. Triggering of apoptosis results in a cascade of caspase activation, in which the last caspases to be activated are those that digest cellular substrates resulting in morphological changes and the death of the cell.
To date, 13 mammalian caspases have been identified [21], and the majority have been analyzed for substrate specificity using a combinatorial library of synthetic peptide substrates [22]. While all caspases have a requirement for an aspartate residue in the P1 position of the cleavage site, they can be classified into three subgroups based on different specificities at the P4 site. Group I includes caspase-1, -4, and -5, which optimally cleave the sequence WEXD [21, 22] and are primarily involved in the generation of the inflammatory response and apoptosis due to pathological causes such as ischemic injury [20]. Group II includes caspase-2, -3, and -7, which prefer DEXD as a substrate [21, 22]. Group III includes caspase-6, -8, and -9, which optimally cleave the sequence (L/V)EXD [21, 22]. The murine caspase-11 and -12 have no established human counterparts at this time and their substrate specificities have not been tested, but both can be categorized as group I caspases based on amino acid sequence homology (both; [19, 20, 23]) and function (caspase-11; [20]).
Caspase activity is regulated by the BCL-2 family of proteins [24–28]. At least 15 mammalian BCL-2 family members have been identified and categorized into two subgroups: those that exert anti-apoptotic effects (BCL-2, BCL-W, BCL-XL, A1, MCL-1) and those that are pro-apoptotic (BAX, BAK, BOK, BIK, BLK, HRK, BNIP3, BIM, BAD, BID, BCL-XS). Although anti-apoptotic homologues can form dimers with pro-apoptotic members, it is controversial whether or not dimerization is required for activity. What is clear, however, is that in most cases the ratio of pro-apoptotic to anti-apoptotic BCL-2 homologues within a cell determines whether the cell will live or die [25, 29].
The list of caspases and BCL-2 family members is continually expanding, and there may be some redundancy of function in both groups. On the other hand, specific caspases and BCL-2 homologues may be utilized differently in different cell types or in response to different triggers [30, 31]. Because the preimplantation embryo develops in a unique, maternal environment, it is of great interest to determine whether the mechanism of cell death utilized by preimplantation embryos is similar to that of other tissues or whether it is highly specialized. Therefore, we have begun to investigate which genes and proteins of the caspase and BCL-2 families are expressed in preimplantation embryos at different stages, as a starting point for further investigations into the precise causes and mechanisms of apoptosis in preimplantation embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All investigations were performed in accordance with the Guide for Care and Use of Laboratory Animals (National Academy of Science, 1996). Female C57BL/6 mice were superovulated with 5 IU eCG (Sigma Chemical Co., St. Louis, MO) at the ninth hour of the light cycle, followed by 10 IU hCG (Sigma) 48 h later. Superovulated females were mated with single males for the collection of embryos but left unmated for the collection of oocytes. Mice were provided with food (NIH31M) and water ad libitum and kept on a 14L:10D cycle (lights-on at 0400 h Eastern Standard Time).
Oocyte and Embryo Collection
Oocytes were collected at 18 h post-hCG. Preimplantation embryos were collected at approximately 18, 42, 66, or 90 h post-hCG for the collection of 1-cell, 2-cell, 8-cell, or blastocyst-stage embryos, respectively. Oocytes and embryos were collected in Whitten-Biggers medium [32] under gas (5% CO2:5% O2:90% N2). Oocytes and 1-cell embryos were treated with 0.3 mg/ml hyaluronidase (Sigma; 300 IU/mg) to remove cumulus cells and sperm.
TUNEL Assay
C57BL/6 embryos were collected at 89 h post-hCG (blastocyst stage), grouped by morphology, and subjected to the TUNEL assay using a kit from PharMingen (San Diego, CA). The embryos were fixed in 1% paraformaldehyde in PBS plus 1% BSA (PBSA) for 15 min at room temperature, washed twice in PBSA, and permeabilized for 5 min in 0.01% Triton X-100/PBSA. The embryos were then washed three times in PBSA and incubated at 37°C, 7% CO2 for 1 h in 50 µl of staining solution containing dUTP-fluorescein isothiocyanate (FITC), terminal deoxynucleotidyl transferase (TdT) enzyme, and reaction buffer. After washing three times in rinse buffer, the embryos were incubated for 30 min at room temperature in propidium iodide/RNase and then rinsed again in PBSA. The embryos were then mounted in 10 µl of PBSA on a microscope slide and squashed gently with a glass coverslip. The embryos were observed immediately using an Olympus (Tokyo, Japan) microscope at x400 magnification under blue light. Photographs were taken using 1600 ASA color film.
Isolation of RNA
Oocytes and embryos were collected, washed three times in PBSA, and then transferred to a 0.6-ml tube in as small a volume as possible. One hundred and sixty cell equivalents were put into each tube, i.e., 160 oocytes or zygotes, 80 2-cell embryos, 20 8-cell embryos, or 5 32-cell blastocysts. The oocytes or embryos were then immediately lysed by adding 100 µl denaturing solution (prepared according to the protocol for the Micro-RNA Isolation Kit [Stratagene, La Jolla, CA]) and vortexing. The samples were spun down briefly and then frozen at -20°C prior to RNA purification. Just prior to RNA isolation, 1 x 106 copies of the synthetic RNA pAW109 (Perkin-Elmer, Foster City, CA) was added to each sample to control for differences in the efficiency of RNA purification and reverse transcription (RT). Total RNA was purified using the Micro-RNA Isolation Kit (Stratagene) according to the manufacturer's protocol, using glycogen as a carrier during precipitation of the RNA. Purified RNAs were immediately converted to cDNA as described below.
RT
Each precipitated, dried RNA sample was resuspended in 12 µl solution consisting of 10.75 µl nuclease-free water (Promega, Madison, WI), 0.2 µl 0.1 M dithiothreitol (Gibco BRL, Gaithersburg, MD), 1 µl 50 µM random hexamers (Gibco BRL), and 0.05 µl (40 U/µl) RNAsin (Promega), prepared as a master mix for all samples. Each sample was vortexed and spun down briefly, then overlaid with 50 µl sterile mineral oil. Hexamers were annealed to the RNA by heating the samples to 70°C for 6 min followed by a 1-min incubation at 25°C. Eight microliters of a solution consisting of 4 µl 25 mM MgCl2 (Perkin Elmer), 2 µl 10-strength polymerase chain reaction (PCR) buffer (Perkin Elmer), 0.4 µl 100 mM dNTPs (Gibco BRL), 0.5 µl (40 U/µl) RNAsin, 0.6 µl nuclease-free water, and 0.5 µl (200 U/µl) Moloney murine leukemia virus reverse transcriptase (Gibco BRL), prepared as a master mix, was then added to each sample; and the sample was mixed by pipetting up and down. The tubes were incubated at 37°C for 3 h, then heated to 99°C for 5 min to terminate the reaction.
PCR
PCR primers were designed using Oligo 5.0 software from National Biosciences, Inc. (Plymouth, MN), based on published sequences obtained from GenBank [23, 29, 33–42]; see Tables 1 and 2. Primers were designed to span introns when intron-exon sequence data were available. In some cases, no data were available on exon boundaries. In these cases, amplification was performed side by side on both cDNA and genomic DNA to test whether products of different size were obtained as an indication that primers in different exons had been chosen. In all cases the primers produced PCR products of the correct size that yielded the expected fragments upon restriction enzyme digestion.
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One twentieth of each sample was amplified using primers for the control RNA, pAW109. For assessment of the expression of caspase and BCL-2 family members, the cDNA equivalent of 16 cells was used in each reaction.
Caspase cDNAs were amplified in 1.5 mM MgCl2, 0.2 mM each dNTP, single-strength PCR buffer, 1 µM each gene-specific primer, and 1.25 U Taq polymerase for 40 cycles, with the following profile: 96°C 1 min for 1 cycle; 96°C 1 min, 53–58°C (see below) 1 min, 72°C 2 min, for 2 cycles; 94°C 1 min, 53–58°C 1 min, 72°C 2 min, for 38 cycles; 72°C 5 min for 1 cycle. Annealing temperatures were 53°C for Caspase-1, Caspase-11, and Caspase-12; 54°C for Caspase-2, Caspase-3, Caspase-7, and pAW109; 58°C for Caspase-6. As a positive control, each transcript was also amplified from mouse lung cDNA, equivalent to 1 µg input RNA, prepared as previously described.
BCL-2 family cDNAs were amplified in 1.5 mM MgCl2, 0.2 mM each dNTP, single-strength PCR buffer, 0.2 µM each gene-specific primer, and 1.25 U Taq polymerase for 40 cycles, with the following profile: 96°C 1 min for 1 cycle; 96°C 1 min, 59°C 1 min, 72°C 15–25 sec, for 2 cycles; 94°C 1 min, 59°C 1 min, 72°C 15–25 sec (depending on the length of the product), for 18 cycles; 94°C 1 min, 59°C 30 sec, 72°C 15–25 sec plus 1 sec per cycle, for 20 cycles; 72°C 5 min for 1 cycle. As a positive control, each transcript was also amplified from mouse liver cDNA, equivalent to 100 ng input RNA, prepared as previously described.
All reverse transcription-polymerase chain reaction (RT-PCR) assays were performed 2–9 times for each stage of development, and RT-PCR products were visualized by electrophoresis through 6% polyacrylamide minigels followed by ethidium bromide staining.
Assay for Caspase Activity Using Fluorogenic Substrates
The PhiPhiLux kit from OncoImmunin Inc. (College Park, MD) was used to detect the activity of group II caspases in both normal and fragmented zygote-stage embryos and in zygotes treated with staurosporine. A group of normal embryos was incubated in Whitten-Biggers medium containing 1 µM staurosporine at 37°C under 7% CO2 for varying lengths of time (hours to days). Control embryos were incubated in Whitten-Biggers only. The embryos were then transferred to 50 µl Whitten-Biggers containing a synthetic caspase substrate and were incubated for varying lengths of time at 37°C under 7% CO2. After incubation with the fluorogenic substrate, the embryos were washed eight times in PBSA, mounted in 10 µl of PBSA on a slide, and squashed gently with a coverslip. The embryos were immediately photographed using an Olympus microscope at x200 or x400 magnification under green light and 1600 ASA color film. The fluorogenic substrate contains the peptide DEVDGID, which can be cleaved by caspases 2, 3, and 7. The substrate also incorporates a molecule of rhodamine on each side of the cleavage site. The two rhodamines interact as a dimer and produce blue-green fluorescence. Cleavage of the substrate disrupts the interaction between rhodamine moieties and results in enhanced red fluorescence.
Immunofluorescence
C57BL/6 embryos were collected at the zygote and blastocyst stages of development and grouped by morphology. The embryos were washed twice in PBSA and then fixed in 1% paraformaldehyde/PBSA for 15 min at room temperature. After washing three times in PBSA, the embryos were permeabilized by a 5-min incubation in 0.01% Triton X-100/PBSA and washed again in PBSA. To inhibit nonspecific binding of antibody, the embryos were incubated for 30 min in 50 µg/ml of FcBlock (PharMingen) and then immediately transferred to primary antibody solution. The embryos were incubated for 1 h at room temperature in primary antibody (50 µg/ml of either rabbit anti-mouse BCL-2 polyclonal antibody or rabbit anti-mouse BAX polyclonal antibody [Santa Cruz Biotechnology, Santa Cruz, CA]) or in PBSA only (control). After washing three times in PBSA, the embryos were transferred to secondary antibody (goat anti-rabbit polyclonal IgG-FITC [Santa Cruz Biotechnology], diluted 1:100 in PBSA), for a 1-h incubation at room temperature in the dark. The embryos were washed two more times in PBSA and then mounted in 10 µl of PBSA on a microscope slide and squashed gently with a glass coverslip. The embryos were immediately examined using an Olympus microscope at x400 magnification under blue light and photographed with 40-sec exposure times using 1600 ASA color film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mouse embryos can be visually categorized as normal or fragmented by light microscopy (Fig. 1). The TUNEL assay was performed on both normal and fragmented embryos, collected at the blastocyst stage, to determine whether fragmentation was associated with apoptotic nicking of genomic DNA (Fig. 2). This assay utilizes the enzyme terminal deoxynucleotide transferase to add dUTP-FITC to the exposed 3'-hydroxyl ends of DNA that are generated during apoptotic DNA fragmentation but not during necrotic death. Thirty-seven normal and five fragmented blastocyst-stage embryos were subjected to the assay. The results from the TUNEL assay confirmed that the embryos that appeared fragmented upon visual inspection were undergoing a much higher degree of apoptosis than normal blastocysts (previously summarized in Warner et al. [9]).
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Expression of Caspase Family Genes in Oocytes and Preimplantation Embryos
Caspases carry out the apoptotic death of the cell and are constitutively expressed in most cell types [1]. Because we have shown that fragmented embryos are undergoing apoptosis, we expected that preimplantation embryos would express caspases. In work with preimplantation embryos, the amount of tissue available for study is very small. Therefore we began our investigations by using the RT-PCR, an easy, inexpensive method of detecting gene transcripts in very small numbers of cells. The results are shown in Figure 3 and summarized in Table 3. The experiments were performed twice on two separate pools of embryos for each stage of development, with the same results.
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Assay for Caspase Enzymatic Activity
We tested zygotes to see whether caspase activity could be detected using a fluorogenic substrate specific for group II caspases preferring the cleavage signal DEXD (caspases 2, 3, and 7). Normal and fragmented zygote-stage embryos (20 of each) were tested for caspase activity. An additional 20 zygotes were treated with staurosporine and then tested for caspase activity. Typical results are shown in Figure 4. Group II caspase activity was very low in normal zygotes, although high levels of enzymatic activity were detected in the polar bodies. In contrast, fragmented zygote-stage embryos displayed high levels of caspase activity in fragments of all sizes. Normal zygotes treated with 1 µM staurosporine did not display caspase activity at 56 h of incubation, but all were stained brightly positive for caspase activity after 72 h of incubation. Of the 20 staurosporine-treated zygotes, 9 did not undergo fragmentation (Fig. 4), 4 disintegrated into brightly stained fragments (data not shown), and 7 looked like abnormal 2-cell embryos with varying degrees of caspase activity in the blastomeres (data not shown).
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Expression of BCL-2 Family Genes in Oocytes and Preimplantation Embryos
Typical RT-PCR results for members of the BCL-2 family are shown in Figure 5 and summarized in Table 4. The experiments were performed 2–9 times for each stage of development. The cause of the variability in detection of some mRNAs in some experiments (e.g., Bcl-2) is unknown.
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Immunofluorescent Detection of BAX and BCL-2 in Zygotes
We chose to examine BCL-2 and BAX proteins for the following reasons: 1) Bcl-2 and Bax transcripts were detected at all stages of preimplantation development; 2) the proteins encoded by these genes are prototypical of anti-apoptotic (BCL-2) and pro-apoptotic (BAX) members of the BCL-2 family; 3) antisera to detect these proteins are commercially available; and 4) the ratio of BCL-2 to BAX can determine whether a cell lives or dies [29]. Because we had detected caspase activity in zygotes, we assessed normal zygotes for the presence of BCL-2 and BAX protein using immunofluorescence (Fig. 6). The experiment was performed twice for each protein, and approximately 30 embryos were assessed in each experiment. Both BAX and BCL-2 immunofluorescence levels were very high in zygotes compared to control embryos treated with secondary antibody only.
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Immunofluorescent Detection of BAX and BCL-2 in Normal and Fragmented Blastocysts
The immunofluorescence assay was performed twice for each protein, and approximately 30 blastocysts were assessed in each experiment. Typical results are shown in Figure 7, although more embryo-to-embryo variation was observed than was seen in zygotes (Fig. 6). While BCL-2 immunofluorescence was consistently greater in normal blastocysts than in fragmented ones, intensities of BAX staining between normal and fragmented blastocysts were more variable.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transcripts for the group II caspase genes (Caspase-2, -3, and -7) were detected in oocytes but not in zygotes. Embryonic Caspase-2 and -3 transcripts were detected at the 2-cell stage, the point at which most of the embryonic genome is activated in mice (zygotic gene activation) [43]. Interestingly, Caspase-7 transcripts were not detected until the 8-cell stage (4-cell embryos were not examined). A fluorogenic substrate was used to detect the activity of this group of caspases in zygotes (Fig. 4). In normal zygotes, caspase activity could be detected only in the polar body. However, fragmented zygotes displayed high levels of caspase activity. Although zygotes were induced to undergo apoptosis after 72 h of incubation in 1 µM staurosporine, most of the zygotes did not undergo fragmentation. Blastocysts can be induced to undergo apoptosis after 6 h of incubation in only 10 nM staurosporine [2, 3]. Thus, zygotes appear to be more resistant to the effects of staurosporine.
Because group II caspase transcripts cannot be detected in zygotes, it seems probable that fertilization results in the rapid degradation of maternal caspase transcripts. Although stored maternal mRNAs are degraded at different rates, depending on the gene, the mRNA begins to be degraded during the resumption of meiosis 12 h before ovulation and is approximately 90% degraded by the mid-2-cell stage [44, 45]. The caspase activity detected in polar bodies and fragmented zygotes is most likely due to stored maternal caspase proteins. However, we cannot rule out certain other possibilities. First, transcripts for an unidentified DEXD-specific caspase may exist in the zygote. Second, embryonic transcription may be initiated in apoptotic zygotes or in zygotes that are cultured for an extended period of time, despite the presence of staurosporine.
Of the group III caspases, the murine homologue had been cloned only for Caspase-6 when these studies were undertaken. (A sequence for mouse Caspase-8 has now been published [46].) Caspase-6 was found to have a typical pattern of expression, in that the maternal transcript was detected in oocytes but not in zygotes, and the embryonic transcript was expressed from the 2-cell stage onward. It has not yet been determined whether the presence of Caspase-6 transcripts corresponds to the presence of protein.
Caspases can be characterized as "initiators" or "effectors" of the caspase cascade [18–21]. Initiators contain domains that target them to specific triggers. The initiators caspase-8 and -10 contain domains that target them to ligand-binding cell surface receptors. Caspase-1, -2, -4, and -9 contain domains that target them either to an internal initiation complex (apoptosome) or to adaptor molecules that interact with cell surface receptors. In contrast, caspase-3, -6, and -7 must be activated by upstream caspases and act as effectors, cleaving substrates that are integral to cellular structure and function. Our experiments have shown that both initiator (caspase-2) and effector (caspase-3, -6, and -7) caspase transcripts are present in oocytes and preimplantation embryos. Due to lack of sequence data and immunological tools, we were unable to establish whether or not caspases associated only with cell surface death receptors (caspase-8 and -10) are expressed. This remains a goal for future experiments.
Having established the likelihood that caspases are synthesized and can be activated throughout preimplantation development, we turned our attention to the regulation of caspase activity by the BCL-2 family of proteins. Because unregulated caspase activity would be deadly to the embryo, it is logical that BCL-2 family members would also be present at each stage of preimplantation development. Although we found this to be true, transcription of BCL-2 family genes appears to be more variable than transcription of the caspase family genes. Only anti-apoptotic Bcl-w and pro-apoptotic Bak were detected 100% of the time at all stages of development. Pro-apoptotic Bax was detected 100% of the time at each stage with the exception of the 8-cell stage, where it was nonetheless detected with high frequency. Anti-apoptotic Bcl-xL was detected 100% of the time at each stage except for the blastocyst stage, where it was also detected with high frequency. Detection of Bcl-xS, the pro-apoptotic variant of Bcl-x L, varied quite a bit. However, it must be noted that bands for Bcl-xS were very faint at best, and it is likely that the amount of Bcl-xS transcribed in any of the samples was at the limit of detection. Similarly, pro-apoptotic Bad and Bid were most often undetectable, but extremely faint bands were occasionally detected in oocytes for Bad and zygotes for Bid. The significance of these expression patterns remains to be determined, but it is possible that the variation in detection of some transcripts may reflect differences in the health of the embryos that are undetectable to the eye.
It is interesting that the family members with nearly undetectable transcripts (Bad and Bid) represent a subfamily of pro-apoptotic family members that display homology to the prototypical members in only one (BH3) of four conserved domains (BH1-BH4). It is thought that members of this subfamily can exert their effects only via dimerization. Whereas BAD (and other members of this group such as BIK and BIM) can only dimerize with anti-apoptotic BCL-2 homologues, antagonizing their function, BID can also dimerize with BAX and appears to enhance its function [47]. It remains to be seen whether Bad and Bid are induced in apoptotic embryos.
Of the caspases examined, only Caspase-12 transcripts were detected in zygotes. In contrast, seven of eight BCL-2 family transcripts (from seven BCL-2 family genes) were detected in zygotes, perhaps ensuring that the regulatory function is always present. It remains to be seen whether these transcripts are maternal (and therefore more stable than maternal caspase transcripts) or embryonic. Very few embryonic genes are expressed in zygotes [43], but it is possible that BCL-2 family genes are expressed at a very early stage to perform the important functions of keeping the embryo alive or eliminating defective embryos.
To evaluate the presence of BCL-2 family proteins in preimplantation embryos, we used fluorescent antibodies to BAX and BCL-2 to label zygotes and blastocysts. Because we had shown that caspase activity could be detected in fragmented zygotes and could be induced in normal zygotes by lengthy treatment with staurosporine, we assessed whether BAX and/or BCL-2 proteins were present in zygotes (Fig. 6). Very bright staining was observed for both proteins in normal zygotes. Although it is not possible to comment on the ratio of one to the other based on this qualitative study, it is clear that the pro-apoptotic activity of BAX must be held in check by the regulatory activity of BCL-2 and/or other anti-apoptotic elements in normal zygotes, which showed no caspase activity. It would be interesting to use scanning confocal fluorescence microscopy to quantitate and compare the amount of BCL-2 and BAX in normal and fragmented preimplantation embryos.
Next, BCL-2 and BAX expression was compared in normal blastocysts vs. fragmented blastocysts (Fig. 7). Although this assay was also qualitative, it appears that the level of BCL-2 immunofluorescence is higher in normal blastocysts than in those with fragmented blastomeres. Our qualitative results support the model, originally put forward by Oltvai et al. [29], that the ratio of BCL-2 to BAX (or anti-apoptotic family members to pro-apoptotic family members) determines whether a cell will live or die.
In conclusion, we have shown that both normal and apoptotic preimplantation embryos express various members of the caspase and BCL-2 families of genes and proteins. We have shown that although there may be unidentified components of the apoptotic machinery that are utilized only in early embryos, preimplantation embryos clearly utilize many of the same proteins used in other cells to execute and regulate cell death, supporting the idea that the basic mechanism is universal. We have begun to correlate changes in protein expression and activity with activation of apoptosis. Questions that remain include the nature of the signals that trigger an embryo to undergo apoptosis, how the expression of other BCL-2 homologues and caspases changes in apoptotic embryos, and how death can be prevented in a clinical setting.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Carol M. Warner, Department of Biology, 414 Mugar Hall, Northeastern University, 360 Huntington Ave., Boston, MA 02115. FAX: 617 373 3724; cmw{at}neu.edu
Accepted: February 17, 1999.
Received: October 27, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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L. Liu, J. R. Trimarchi, and D. L. Keefe Haploidy but Not Parthenogenetic Activation Leads to Increased Incidence of Apoptosis in Mouse Embryos Biol Reprod, January 1, 2002; 66(1): 204 - 210. [Abstract] [Full Text]
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 J. K. Pru and J. L. Tilly Programmed Cell Death in the Ovary: Insights and Future Prospects Using Genetic Technologies Mol. Endocrinol., June 1, 2001; 15(6): 845 - 853. [Abstract] [Full Text] [PDF]
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T. Matikainen, G. I. Perez, T. S. Zheng, T. R. Kluzak, B. R. Rueda, R. A. Flavell, and J. L. Tilly Caspase-3 Gene Knockout Defines Cell Lineage Specificity for Programmed Cell Death Signaling in the Ovary Endocrinology, June 1, 2001; 142(6): 2468 - 2480. [Abstract] [Full Text] [PDF]
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K. Kawamura, J. Fukuda, H. Kodama, J. Kumagai, A. Kumagai, and T. Tanaka Expression of Fas and Fas ligand mRNA in rat and human preimplantation embryos Mol. Hum. Reprod., May 1, 2001; 7(5): 431 - 436. [Abstract] [Full Text] [PDF]
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L. Hinck, P. Van Der Smissen, M. Heusterpreute, I. Donnay, R. De Hertogh, and S. Pampfer Identification of Caspase-3 and Caspase-Activated Deoxyribonuclease in Rat Blastocysts and Their Implication in the Induction of Chromatin Degradation (but Not Nuclear Fragmentation) by High Glucose Biol Reprod, February 1, 2001; 64(2): 555 - 562. [Abstract] [Full Text]
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 S. Pampfer, S. Cordi, I. Vanderheyden, P. Van Der Smissen, P. J. Courtoy, A. Van Cauwenberge, H. Alexandre, I. Donnay, and R. De Hertogh Expression and Role of Bcl-2 in Rat Blastocysts Exposed to High D-Glucose Diabetes, January 1, 2001; 50(1): 143 - 149. [Abstract] [Full Text]
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J. R. Trimarchi, L. Liu, P. J.S. Smith, and D. L. Keefe Noninvasive Measurement of Potassium Efflux as an Early Indicator of Cell Death in Mouse Embryos Biol Reprod, September 1, 2000; 63(3): 851 - 857. [Abstract] [Full Text]
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K. E. Latham, B. Patel, F. D. M. Bautista, and S. M. Hawes Effects of X Chromosome Number and Parental Origin on X-Linked Gene Expression in Preimplantation Mouse Embryos Biol Reprod, July 1, 2000; 63(1): 64 - 73. [Abstract] [Full Text]
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L. Liu, J. R. Trimarchi, and D. L. Keefe Involvement of Mitochondria in Oxidative Stress-Induced Cell Death in Mouse Zygotes Biol Reprod, June 1, 2000; 62(6): 1745 - 1753. [Abstract] [Full Text]
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A. C. Gordo, H. Wu, C. L. He, and R. A. Fissore Injection of Sperm Cytosolic Factor Into Mouse Metaphase II Oocytes Induces Different Developmental Fates According to the Frequency of [Ca2+]i Oscillations and Oocyte Age Biol Reprod, May 1, 2000; 62(5): 1370 - 1379. [Abstract] [Full Text]
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Q. Wang and K. E. Latham Translation of Maternal Messenger Ribonucleic Acids Encoding Transcription Factors During Genome Activation in Early Mouse Embryos Biol Reprod, April 1, 2000; 62(4): 969 - 978. [Abstract] [Full Text]
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 A. Voss, T Thomas, P Petrou, K Anastassiadis, H Scholer, and P Gruss Taube nuss is a novel gene essential for the survival of pluripotent cells of early mouse embryos Development, January 12, 2000; 127(24): 5449 - 5461. [Abstract] [PDF]
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