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-Deficient Mouse Blastocysts1
a Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) reduces the incidence of apoptosis in mouse blastocysts that develop in vitro but does not result in an increase in cell number or the incidence of development to the blastocyst stage. Thus, TGF may function as a cell survival factor in the preimplantation mouse embryo. To extend these studies, we have now examined the development of TGF-deficient preimplantation embryos in vitro and in vivo in TGF-deficient mothers. We found that in both instances the incidence of apoptosis is dramatically increased in the TGF-deficient blastocysts and that this increase is essentially restricted to the cells of the inner cell mass when the embryos develop in vivo but extends to the trophectoderm cells for embryos that develop in vitro. The absence of endogenous TGF has little effect on the incidence of development to the blastocyst stage and cell number, cell lineage allocation, blastocoel volume, and the timing and incidence of hatching in these blastocysts, when compared to wild-type embryos. These results buttress our previous suggestion that TGF functions as a cell survival factor in the preimplantation mouse embryo.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell division, differentiation, and death in embryos, as in adults, are likely to be regulated by peptide growth factors. Embryos express a variety of growth factor receptors and ligands, and exogenous growth factors can modulate many aspects of embryo development [8–10]. For example, the embryo expresses transforming growth factor alpha (TGF) and its receptor, the epidermal growth factor (EGF) receptor (EGFR), but not EGF, throughout preimplantation development ([11–13]; unpublished results). In the blastocyst, TGF and EGFR are expressed in both the ICM and TE cells [12, 14, 15]. When embryos are cultured under suboptimal conditions, exogenous TGF can increase the rate at which embryos develop, the fraction that reach the blastocyst stage, and the final cell number [16]. Exogenous TGF can also stimulate the rate of blastocoel expansion [13, 17], as well as the rate of DNA [13] and protein [13, 14] synthesis, the secretion of proteins by the blastocyst [18] and the pattern of gene expression [19]. These experiments implicate EGFR signaling in preimplantation development. A critical role for EGFR signaling in ICM development is indicated by the fact that one phenotype observed after ablation of the EGFR gene is periimplantation lethality on Day 6.5 associated with failure of the ICM to develop [20]. There is still little direct evidence, however, for a role for endogenous TGF in preimplantation development, although TGF antisense oligonucleotides do decrease the rate of blastocoel fluid accumulation [13].
Continuous signaling from survival factors is required to prevent all nucleated mammalian cells that have been studied from undergoing the default cell fate of PCD [21, 22]. When cultured at low density (which dilutes out secreted survival factors) or exposed to the protein kinase inhibitor staurosporine (which is presumed to block all extracellular signaling), these somatic cells undergo apoptosis ([22, 23], reviewed in [24]). In many cell types, peptide growth factors can act as survival factors [25]; for example, both TGF [26–28] and EGFR ([29], references in [30]) have recently been implicated in the regulation of apoptosis. Cell death in the blastocyst may also be regulated by extracellular survival factors, since TE or ICM cells undergo apoptosis when exposed to staurosporine [22]. In contrast, early cleavage-stage blastomeres do not die when they are cultured as single cells, and are less sensitive to staurosporine, suggesting that while blastomeres have the cell death machinery, they may not have an absolute requirement for cell survival factors [22].
Results of our recent experiments are consistent with a role for growth factors, and TGF in particular, as cell survival factors in the preimplantation mouse embryo. For example, when embryos are cultured singly in individual drops from the 2-cell stage to the blastocyst stage, apoptosis in the ICM is increased by up to 3-fold, compared to apoptosis in control blastocysts that have developed in vivo [30], suggesting that factors present in the maternal tract may act as survival factors. These factors may also be secreted by the embryos themselves, since the incidence of apoptosis is reduced after culture of 2-cell embryos to blastocysts at high density (30 embryos per drop). Moreover, inclusion of 0.1 pM TGF reduces the incidence of apoptosis in the ICM of blastocysts that develop from singly cultured 2-cell embryos [30]. In toto, these data suggest for the first time that embryo-derived factors may have a role as survival factors that regulate cell death during preimplantation development, and that TGF may be one of these factors. Nevertheless, an absolute requirement of ICM cells for survival factors such as TGF is difficult to assess from these experiments, since both the ICM and the TE express TGF [15] and the ICM is maintained in the microenvironment of the blastocoel, which may contain soluble secreted TGF. In fact, when ICMs are removed from the blastocoel by immunosurgery and cultured singly for 24 h, they show a dramatic increase in the incidence of apoptosis that is partially reduced by adding exogenous TGF to the medium [30]. Thus, factors such as TGF present in the blastocoel may act as cell survival factors for the ICM. In addition, since TGF and EGFR are coexpressed in the ICM [14], juxtacrine signaling between neighboring cells [31] may also promote survival.
In order to address the question of the requirement of ICM cells for endogenous TGF as a survival factor and to explore further other possible roles of this growth factor, we examined preimplantation development in mice lacking a functional TGF gene [32]. TGF-deficient (i.e., -/-) mice show a relatively mild phenotype, including skin architecture, hair follicle, and eyelid abnormalities [32, 33], and are fertile and healthy. Thus, TGF is not essential for any stage of development, including preimplantation development, since TGF -/- embryos can develop in a TGF -/- mother. Compensation by other members of the EGF family present in the maternal tract or expressed by the embryo (e.g., EGF, heparin-binding EGF, amphiregulin [14, 34, 35], or other growth factors, probably accounts for this result. In an attempt to clarify the role of TGF in preimplantation development, we conducted a detailed analysis of development to the blastocyst stage in TGF -/- embryos that developed either in vivo, i.e., in the presence of other maternal tract factors, or in vitro, i.e., in the absence of those factors. Furthermore, in vitro embryos were cultured singly in drops in order to dilute out and thereby effectively prevent the function of other endogenous, secreted growth factors [10, 16, 30]. In these studies we employed a method for analyzing cell proliferation, apoptosis (identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL]), and cell allocation to the ICM and TE in blastocysts using confocal microscopy followed by three-dimensional image reconstruction. In general, we found that TGF -/- embryos develop into morphologically normal blastocysts in vitro or in vivo, with a similar size, total cell numbers, mitotic index, and allocation to ICM and TE lineages, as compared to wild-type control embryos that express TGF. TGF -/- blastocysts developing in vitro or in vivo, however, show a much higher incidence of apoptosis in the ICM, compared to wild-type control embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TGF -/- [33] and control B6129 (TGF +/+) background strain male and female mice were obtained from the Jackson Laboratories (Bar Harbor, ME). TGF -/- animals were easily distinguished from TGF +/+ animals by virtue of their abnormal hair patterning and whiskers [33]. Two-cell-stage embryos were recovered on Day 2.0 (48–50 h post-hCG) and blastocysts on Day 4.0 (96–98 h post-hCG) from the oviducts and uteri, respectively, of superovulated -/- and +/+ females mated to TGF -/- and +/+ males, respectively, as previously described [17, 31]. The flushing medium was bicarbonate-free minimal essential medium (Earle's salts) containing pyruvate (100 µg/ml), gentamicin (10 µg/ml), polyvinylpyrrolidone (PVP; 3 mg/ml), and 25 mM HEPES, pH 7.2.
Embryo Culture
TGF +/+ and -/- 2-cell embryos were cultured to the blastocyst stage singly in 25-µl drops of KSOM medium [36] containing essential and nonessential amino acids (Gibco BRL, Grand Island, NY) and fatty acid-free BSA (1 mg/ml; Sigma, St. Louis, MO) (designated KSOM+AA [37]) under mineral oil (Sigma) in a humidified atmosphere of 5% CO2:5% O2:90% N2 at 37°C, as described previously [30]. The embryos were transferred to fresh drops of medium every 24 h, at which time the developmental stage was also recorded. The cultures were terminated on Day 5.0 (114 h post-hCG) to ensure that nearly all embryos had formed blastocysts. In order to eliminate sampling biases, all embryos possessing a blastocoel were selected for labeling.
TUNEL and Propidium Iodide (PI) Labeling
TUNEL and PI labeling were carried out as described previously [30]. Briefly, zona pellucida-intact embryos were washed several times in PBS (pH 7.4) containing PVP (1 mg/ml), fixed in 3.7% paraformaldehyde in PBS for 1 h at room temperature or overnight at 4°C, and permeabilized in 0.5% Triton X-100 (Sigma) for 1 h at room temperature. The embryos were then washed twice in PBS/PVP, and next incubated in fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase (TUNEL reagents; Boehringer Mannheim, Indianapolis, IN) for 1 h at 37°C in the dark. After TUNEL, embryos were counterstained with PI plus RNase A (50 µg/ml of each) for 60 min at room temperature to label all nuclei. Embryos were washed extensively and mounted with slight coverslip compression in VectaShield anti-bleaching solution (Vector Labs., Burlingame, CA). Great care was taken to ensure that the blastocysts did not collapse during immersion into VectaShield and that coverslip compression was sufficient to immobilize the blastocyst but not cause it to collapse. Slides were sealed with nail varnish and stored at 4°C in the dark for up to several days until confocal analysis.
Confocal Microscopy and Analysis
Fluorescence was detected using a Leica TCS 4D laser-scanning confocal microscope (Leica, Milton Keynes, Bucks, UK). Each blastocyst was scanned in two channels, red to detect PI and green to detect the TUNEL fluorescein isothiocyanate (FITC)-label. A complete Z series was performed for each blastocyst, consisting of at least 20 optical sections at approximately 3- to 4-µm intervals, in order to ensure that each nucleus in the blastocyst was sampled. Scanware software (Leica) was used to generate a red/green three-dimensional reconstruction of each blastocyst that was analyzed at angles from +25° to -25°. These reconstructions were displayed simultaneously as rotating movies in each of the two channels, which allowed counting and analysis of nuclei in each compartment of the blastocyst. The use of this method to count nuclei was validated by subsequently squashing and disaggregating some of the blastocysts and then counting spread nuclei by epifluorescence microscopy. After analysis, the confocal images were stored as the original Z series.
Each blastocyst was analyzed for total number of nuclei, number of mitotic nuclei, and number of TUNEL-labeled nuclei in the ICM, polar TE, mural TE, and blastocoel. TE nuclei were distinguished from ICM nuclei by a combination of position and morphology. TE nuclei were smaller, more intensely stained, and slightly flattened against the zona pellucida, whereas ICM nuclei were larger, rounded, and less intensely stained. The position of the outer TE nuclei was easy to assign by rotating the image of the blastocyst to different angles about its x or y-axis. Polar TE nuclei were distinguished from mural TE nuclei by their position immediately overlying, and adjacent to, ICM nuclei. The optical sectioning capability of this method allows cells to be assigned to each compartment on the basis of a three-dimensional reconstruction of the intact blastocyst. In this respect, the method is similar to that in the early studies describing cell allocation and death in serially sectioned blastocysts ([2, 3] and references in [4] and [38]). The confocal method, however, is far less labor-intensive and permits analysis of fluorescent markers. The confocal method also differs from the commonly used differential ICM/TE labeling method [30, 38, 39], in which TE and ICM nuclei are discriminated functionally; TE cells are partially lysed and labeled with both PI and bisbenzamide, whereas the ICM cells remaining intact within the TE permeability seal are labeled with only the membrane-permeable bisbenzamide. Nuclei in each compartment are counted by disaggregating the blastocyst, and dead cells can be identified by their fragmented nuclear morphology. In comparison, the advantage of the confocal method is that it preserves whole-mount morphology, which makes it easier to identify morphological features such as dead cells loose in the blastocoel. Apoptotic cells are also more readily identified by TUNEL, which is a biochemical criterion of apoptosis, rather than the previously used morphological criterion of nuclear fragmentation. The major limitations of the confocal method are that it can be applied only to relatively early blastocysts (< 100 cells) and that it requires extensive confocal time.
The approximate volume of each blastocyst was calculated from the depth (measured as the difference between the top and bottom of the Z series) and the diameter (calculated from the reconstructed image).
Statistical Analysis
Differences between percentages of embryos developing in culture were compared by chi-square analysis with Yates' correction, or Fisher's Exact Test. Differences between +/+ and -/- embryos in each culture were tested for statistical significance using an unpaired t-test for cell numbers, and the Mann-Whitney U test for dead cell numbers and mitotic and dead cell indices. Statview 4.0 statistical package for the Macintosh was used for all analyses. Blastocysts for each group were recovered on at least two and usually three separate occasions and from at least four different females for each experiment, in order to minimize maternal effects. All labeled blastocyst slides were coded, and the analysis by confocal microscopy and scoring were performed blind.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-/- Mice
The breeding performance of the TGF -/- colony, in terms of frequency and size of litter production, was poor and decreased with the age of the animals. This may have been a function of the B6129 strain, rather than the TGF -/- phenotype, since the breeding performance of the B6129 TGF +/+ animals also decreased with age. Although TGF -/- and +/+ mice could be superovulated and after mating yielded similar numbers of blastocysts on Day 4, the yield of 2-cell-stage embryos on Day 2 in TGF -/- mice was only half that of +/+ mice (7.9 per -/- female, compared to 17.7 per +/+ female). This lower yield could reflect a role for TGF in follicle development and ovulation.
Gross Phenotype of TGF -/- Preimplantation Embryos
Since wild-type mice express TGF in the oocyte, egg, and preimplantation embryo ([11, 13–15] and unpublished results), we mated fertile homozygous TGF -/- males and females to examine the formation of blastocysts in the complete absence of TGF from any source, including maternal pools in the oocyte, the reproductive tract, and the developing embryo itself. In addition, since both preimplantation embryos and the maternal tract express other growth factors that could also act as survival factors, we allowed TGF -/- embryos to develop to the blastocyst stage in vivo (i.e., in the presence of other compensating maternal tract factors) or cultured them singly in vitro from the 2-cell stage (i.e., in the absence of other maternal tract factors or those derived from other embryos).
Two-cell embryos flushed on Day 2 from TGF +/+ and -/- females were cultured singly to the blastocyst stage in KSOM+AA medium. We chose this medium because KSOM+AA fosters the best rates of development in vitro reported to date and hence culture in KSOM+AA should minimize the effect of suboptimal culture conditions [30, 37]. Although TGF -/- animals yielded fewer 2-cell embryos than the +/+ animals, these -/- 2-cell embryos developed normally to the blastocyst stage. There were no gross differences between TGF +/+ and -/- embryos in the timing of embryo development in vitro to the blastocyst stage, the percentage of embryos that reached the blastocyst stage, or the morphological appearance and final expanded volume of these blastocysts (Table 1). While the incidence of hatching appeared reduced, this difference was not statistically significant (Table 1). TGF -/- or +/+ blastocysts that developed in vivo and were flushed from the reproductive tract on Day 4 also showed no significant differences with respect to gross morphological appearance or blastocyst volume (Table 1).
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Cell Proliferation and Allocation in TGF +/+ and -/- Embryos
We examined qualitative and quantitative indices of cell proliferation in mouse blastocysts by fixing and permeabilizing individual blastocysts and staining with PI to label all nuclei (Figs. 1 and 2). Blastocysts were optically sectioned using confocal microscopy, and computer software was used to generate three-dimensional reconstructed images (Fig. 1, B, C, and E, and Fig. 2, A–F). These images were displayed as rotating movies that permitted discrimination between each compartment of the blastocyst: ICM, polar and mural TE, and the blastocoel. Total nuclear number and mitotic index were scored in each compartment, as well as apoptotic cells, as assessed by TUNEL labeling.
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There were no differences between TGF +/+ and -/- blastocysts in terms of total cell number, or of the number of cells in the ICM and polar or mural TE, for either in vivo or in vitro developed embryos (Table 1). TGF -/- blastocysts that developed in vivo showed a slight decrease in the percentage of cells allocated to the ICM when compared to +/+ embryos (Table 1). The significance of this, however, is questionable since these embryos can develop into live mice and since a similar decrease was not seen in in vitro blastocysts. In addition, there was a 4-fold decrease in mitotic index in the ICM of TGF -/- blastocysts that developed in vivo, but a similar decrease was not seen in those that developed in vitro (Table 1). On the other hand, the mitotic index of mural TE was not different for blastocysts that developed in vivo but was slightly reduced in TGF -/- blastocysts that developed in vitro (Table 1). This might be a result of the absence of secreted mitogenic growth factors in drops containing only one embryo, combined with the absence of endogenous TGF.
Cell Death by Apoptosis in TGF +/+ and -/- Embryos
Cell death in all four categories of blastocysts (TGF +/+ and -/- embryos that developed in vitro or in vivo) was seen predominantly in the ICM but occasionally in all compartments of the blastocyst, including polar and mural TE, as well as in the blastocoel (as cells floating loose) (Table 1, Figs. 1–4). Apoptotic cells that appeared in the blastocoel were scored separately, although they may have been derived from the ICM. Similarly, apoptotic cells that appeared to be present in the polar TE layer were scored as polar TE but may have originated in the ICM. Apoptotic nuclei were only occasionally observed trapped between the layer of healthy TE cells and the zona pellucida (e.g., Fig. 2C). Blastocysts that developed in vitro showed an increase in cell death number and incidence compared to their in vivo counterparts, for both TGF +/+ and -/- embryos (Figs. 2 and 3).
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The major factor affecting cell death number and incidence in blastocysts appeared to be the presence or absence of the TGF gene. TGF -/- blastocysts showed a 2- to 3-fold increase in the total number and overall incidence of apoptotic nuclei identified by TUNEL, for both in vivo and in vitro developing embryos, when compared to TGF +/+ control blastocysts (Figs. 3 and 4). TGF -/- blastocysts that developed in vivo showed this increase solely in the ICM, with only the occasional dead cell present in the polar or mural TE or loose in the blastocoel (Fig. 2C), the incidence of which was not significantly increased in TGF -/- blastocysts. Blastocysts that developed in vitro also showed a large increase in ICM cell death in TGF -/- embryos, when compared to TGF +/+ embryos (Fig. 3). These blastocysts, however, also showed a large increase in the incidence of dead cells outside the ICM (Figs. 2, D–F, and 4). TGF -/- blastocysts that developed in vitro averaged one dead cell in each of the polar and mural TE, and blastocoel compartments, such that 50% of their dead cells were located outside of the ICM (Fig. 2, D–F). This represented an approximately 2- to 3-fold increase in the incidence of cell death outside the ICM, when compared to TGF +/+ embryos that developed in vitro, and a 4- to 5-fold increase compared to TGF +/+ blastocysts that developed in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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functions as a cell survival factor in the preimplantation mouse embryo [30], since blastocysts from TGF -/- mice showed an increased incidence of apoptosis. The absence of TGF had relatively little effect on other aspects of preimplantation development.
In general, when compared to wild-type embryos, TGF -/- embryos that developed in either TGF -/- mothers or in vitro formed morphologically normal blastocysts at a similar time and at a similar incidence. The final volume of the blastocoel and ability to hatch from the zona pellucida were not significantly different from those characteristics in wild-type embryos. Moreover, these blastocysts showed normal cell proliferation compared to that of their wild-type counterparts and contained a similar total number of cells and a relatively normal allocation to the ICM and TE. TGF may also serve as a mitogen since the TGF -/- blastocysts that developed in vivo have a greatly reduced mitotic index in the ICM. In combination with the increased incidence of apoptosis, this could account for the slightly reduced allocation to the ICM in these blastocysts. These results are generally in agreement with our previous findings that exogenous TGF added to wild-type TGF-expressing CF-1 embryos does not affect the timing or incidence of blastocyst formation, blastocyst volume or final cell number, mitotic index, or allocation to the ICM or TE [17, 30]. Another recent study has also shown that exogenous TGF does not increase final cell number in blastocysts of CD-1 mice; however, this study did report that exogenous TGF stimulates the incidence of blastocyst formation [35]. The difference between this result and our findings [30] may reflect differences in mouse strain or experimental conditions. Although TGF can stimulate blastocoel expansion [13, 17, 35], it clearly is not required for blastocoel expansion, since the final volume of the fully expanded blastocyst is achieved in the absence of TGF function. In general, although it is clear that the addition of exogenous TGF to embryos can stimulate a number of functions (see Introduction), presumably via EGFR signaling, endogenous TGF itself does not appear to be absolutely required.
TGF -/- blastocysts that develop in vivo in TGF -/- mothers have normal developmental competence (i.e., the animals are fertile [32, 33] and the present study) and appear to be morphologically normal (the present study). This is not surprising, since other factors present in the maternal tract could compensate for TGF function. On the other hand, the robust development of TGF -/- 2-cell embryos cultured singly to the blastocyst stage may be construed as surprising, since they develop under conditions in which maternal factors cannot compensate for TGF function and in which embryo-derived factors secreted into the medium are presumably diluted and ineffective [10, 30]. This suggests that either TGF has no function in preimplantation development (but see below), or that other embryo-derived factors retained by the embryo (perhaps secreted into the blastocoel) compensate for TGF function. For example, preimplantation mouse embryos express amphiregulin, which can stimulate blastocyst development and cell proliferation, presumably by binding to and activating the EGFR and/or by localizing to the nucleus [35].
The most striking phenotype observed for the TGF -/- embryos that develop either in vivo in a TGF -/- mother or in vitro is a 2- to 3-fold increase in the number of apoptotic cells, which are mostly confined to the ICM. This result is consistent with our previous observation that exogenous TGF can reduce the incidence of apoptosis in blastocysts that develop from singly cultured 2-cell embryos and confirms our suggestion that TGF functions as a cell survival factor in the preimplantation mouse embryo [30]. The incidence of dead cells outside of the ICM is low in both TGF -/- and +/+ blastocysts that develop in vivo, i.e., apoptosis is essentially restricted to the ICM. In contrast, TGF -/- blastocysts that develop in vitro also contain significantly greater numbers of dead cells in the polar and mural TE, as well as loose in the blastocoel. Dead cells loose in the blastocoel have been described previously for other mouse strains and species ([2, 3, 30] and references in [4]). These may be derived from the TE, or more likely the ICM, after loss of intercellular contacts and extrusion from the ICM. Alternatively, they may be cells that have become apoptotic earlier in development and were not phagocytosed because they lacked cell surface markers, or because the surrounding blastomeres had not yet developed phagocytic capability. These would therefore be excluded during blastocyst formation, as has been suggested for the human embryo [4, 40]. TGF -/- embryos, however, do not show any obvious signs of apoptosis at earlier stages of development in vitro.
The incidence of cell death in both TGF +/+ and -/- embryos that develop in vitro is greater than in their counterparts that develop in vivo. This is similar to our previous observations in CF-1 blastocysts and probably reflects the ability of maternally derived factors to function as cell survival factors and/or the reduced capacity of cultured embryos to express such factors [30]. In particular, the increased incidence of apoptosis in the TE cells of these embryos that develop in vitro may also suggest that maternal tract factors (perhaps including TGF) act as cell survival factors for TE cells. Thus, during normal development, endogenous embryo-derived TGF may regulate cell death in the ICM and the TE, perhaps via autocrine or juxtacrine signaling, whereas maternally derived cell survival factors present in the reproductive tract may regulate cell death in the TE cells, which are located on the outside of the embryo and are exposed to the maternal environment. This would explain why embryos developing in vivo in the total absence of TGF but in the presence of other maternal tract factors show increased cell death in the ICM but not in the TE, whereas embryos developing in vitro in the absence of both TGF and other maternal tract factors show the increase in all compartments of the blastocyst.
The question of whether or not the ICM has an absolute requirement for cell survival factors remains unresolved. While there clearly is no absolute requirement for TGF, there may be such a requirement for other embryonic survival factors such as other EGFR ligands. One of the EGFR null mutant phenotypes is periimplantation death around Day 6.5 that is due to failure of ICM development [20]. Thus, on at least one genetic background, EGFR signaling is required for ICM development, perhaps to ensure cell survival. Although there is an increase in cell death in the ICM in the absence of TGF, other factors that activate the EGFR, such as amphiregulin, may compensate for the absence of TGF function and thereby sustain ICM cell survival to a degree sufficient for continued embryo development. Thus, although TGF appears to function as a cell survival factor in the preimplantation mouse embryo, its absence is compensated by other factors. Results presented in this study constitute another example of compensatory mechanisms that function during development. Moreover, our results highlight the growing realization that while such mutant mice may not manifest grossly apparent phenotypic changes, close examination will most likely reveal more subtle phenotypic changes that suggest functions not previously apparent or appreciated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Richard Schultz, Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018. FAX: (215) 898-8780; rschultz{at}mail.sas.upenn.edu
3 Current address: Department of Reproductive Medicine, St. Mary's Hospital, Manchester M13 OJH, UK.
Accepted: February 27, 1998.
Received: December 29, 1997.
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A.V. Makarevich and M. Markkula Apoptosis and Cell Proliferation Potential of Bovine Embryos Stimulated with Insulin-Like Growth Factor I During In Vitro Maturation and Culture Biol Reprod, February 1, 2002; 66(2): 386 - 392. [Abstract] [Full Text] [PDF]
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