Mitogenic growth factors are thought to play key regulatory roles in preimplantation embryo development by promoting nuclear and cytoplasmic maturation, increasing fecundity and rates of fertilization, optimizing cellular cleavage, and synchronizing embryonic and maternal maturation. Our results, in the context of our earlier work, strongly support the contention that mammalian embryos are particularly sensitive to the presence of mitogenic growth factors and that adequate levels and appropriately timed expression of these factors are crucial for successful preimplantation embryo development. This necessary role in development indicates that EGF and TGFa expression and in turn EGFR activation are essential for ensuring appropriate cell proliferation and differentiation and thus promote survival and normal preimplantation development.
Our findings are consistent with results that indicate a lower rate of development and a greater incidence of programmed cell death (that is, apoptosis) in IVF and cloned embryos than in vivo-derived embryos. To a variable extent, these earlier observations were dependent on culture conditions, which likely affect gene expression and nuclear reprogramming. Our experimental design controlled for this variable by applying identical culture conditions to all 3 embryo groups. In this way, we could compare and contrast results between fertilized and cloned embryos in order to specifically address the role of mito-genic growth factors in preimplantation embryo development. Continue reading
In the present study we determined that the development of mouse embryos derived by either natural mating or IVF was negatively affected by immunoneutralization of mitogenic growth factors and their receptor. Treatment with immunoneutralizing antibodies to EGF, TGFa, and EGFR decreased the rate of development of 1-cell-stage embryos to blastocysts, reduced the total number of differentiated cells in the ICM and TE, decreased cell size, reduced the ICM:TE ratio, and increased the fragmentation of nuclear DNA (that is, apoptosis). These differences were accentuated, in most cases, when embryos were treated simultaneously with neutralizing antibodies to both EGF and TGFa. In vitro-fer-tilized embryos appeared to be more sensitive than in vivo-de-rived embryos to immunoneutralizing antibodies. Therefore, the negative effect of neutralizing antibodies to mitgoegnic growth factors on the growth and differentiation characteristics of fertilized embryos indicates a direct relationship between the actions of EGF and TGFa and normal development of mouse embryos. Continue reading
Number of cells and differential labeling. As the total number of embryonic cells decreases, the developmental potential of preimplantation mammalian embryos decreases. Compared with that after control antibody treatments, the total number of cells (mean + SE) in in vivo-derived embryos was significantly (P < 0.05) less after treatment with neutralizing antibodies to EGF (68.8 + 0.9 cells), TGFa (67.8 + 1.6 cells), and EGFR (68.0 + 2.2 cells) and combined treatment with antibodies to both EGF and TGFa (59.4 + 0.7 cells; Table 3 and Figure 1 A, D, and G). Similar relative results were observed after treatment of in vitro-fertilized embryos with neutralizing antibodies to EGF (63.8 + 0.7 cells), TGFa (63.0 + 1.2 cells), and EGFR (63.6 + 1.3 cells) and combined treatment with antibodies to both EGF and TGFa (57.4 + 0.5). Cell size was decreased after immunoneutralization treatment (Figure 1 D). Continue reading
Apoptosis. Apoptosis, or programmed cell death, is an important parameter by which to assess the developmental competence of preimplantation mammalian embryos. Apoptotic cells were detected in all groups of embryos after treatment with neutralizing antibodies (Table 2 and Figure 1 B and E). Compared with that after control antibody treatment, the numbers of cells (mean + SE) with apoptotic nuclei among in vivo-derived embryos were significantly (P < 0.05) greater after treatment with neutralizing antibodies to EGF (7.3 + 0.4 cells), TGFa (7.7 + 0.5 cells), and EGFR (7.5 + 0.3 cells) and combined treatment with antibodies to both EGF and TGFa (10.3 + 0.3 cells). Similar relative and statistically significant (P < 0.01) results were observed after treatment of in vitro-fertilized embryos with neutralizing antibodies to EGF (9.4 + 0.3 cells), TGFa (9.6 + 0.4 cells), and EGFR (9.4 + 0.2) and combined treatment with antibodies to both EGF and TGFa (10.8 + 0.5 cells). In both groups of embryos, after treatment with neutralizing antibodies, 92% to 94% of apoptotic cells were in the ICM (Figure 1 B and E). Continue reading
Rate of development and embryo morphology. Compared with those after control antibody treatments, the numbers of blastocysts (mean + standard error) that developed from in vivo-derived 1-cell-stage embryos were significantly (P < 0.05) reduced after treatment with neutralizing antibodies to EGF (27.3 + 0.8 blastocysts), TGFa (26.5 + 0.6 blastocysts), and EGFR (26.3 + 0.7 blastocysts; Table 1). Similar relative results were observed after treatment of in vitro-fertilized embryos with neutralizing antibodies to EGF (22.6 + 0.3 blastocysts), TGFa (21.5 + 0.2 blastocysts), and EGFR (21.2 + 0.6 blastocysts). Compared with that of in vivo-derived embryos, development of in vitro-fertilized embryos was poorer after immunoneutralization. Further, the numbers of blastocysts that developed after combined treatment with neutralizing antibodies to both EGF and TGFa of in vivo-derived embryos (12.6 + 0.6 blastocysts) and in vitro-fertilized embryos (11.8 + 0.4) were significantly (P < 0.01) fewer than after treatment with either control or individual neutralizing antibodies. Continue reading
Number of cells and differential labeling. To determine to what extents the numbers of cells and ICM:TE ratio were affected by treatment with neutralizing antibodies, ICM and TE cells were differentially labeled using PI and bisbenzimide (Hoechst 33258) by using a modification of a previously published protocol. Briefly, blastocysts with intact zona pellucida were incubated in 500 ц1 HEPES-buffered HTF (Specialty Media) containing 1% Triton X100 and 100 |ig/ml PI for 10 s, during which time changes in the color and size of TE cells could be seen microscopically. Blastocysts were transferred immediately into 500 ц1 100% ice-cold ethanol containing 25 |ig/ml bisbenzimide and incubated overnight at 4 C. Fixed and stained blastocysts were mounted on a glass slide in 5 ц1 glycerol and gently flattened with a coverslip, and fluorescence was visualized under a fluorescence microscope. To analyze the total number of cells, cells were visualized using a filter for DAPI (330 to 380 nm), whereas differentially labeled ICM and TE cells were visualized using different filters (460 nm for blue and red fluorescence, and 560 nm for red only). Continue reading
Assays. TUNEL Assay. To determine the amount of apoptosis affecting in vivo-derived, in vitro-fertilized, and ICNI-derived cloned embryos, blastocysts with intact zona pellucida were selected randomly from each group and fixed, permeabilized, and stained as described previously. Briefly, blastocysts were transferred into 50 ц1 of a 1-mg/ml solution of PVP in phosphate buffered saline (PBS; PVP-PBS), and fixed in 4% paraformaldehyde in PBS for 1 h at room temperature or overnight at 4 C.
Activation. Activation was induced by incubation of reconstructed oocytes in 50 ц1 Ca2+-free CZB containing both 10 mM Sr2+ (Sigma, St Louis, MO) and 5 |ig/ml cytochalasin B for 6 h at 37 C under 5% CO2 in air. After activation, reconstructed oocytes were allocated into 2 treatment subgroups of approximately 20 to 25 1-cell-stage embryos each and transferred to culture medium supplemented with either positive-control antibody or no antibody (negative control) as described previously. Continue reading
Donor nuclei. Cumulus cells in approximately 100 ц1 hyal-uronidase solution were transferred into a 1.5-ml microtube containing 1 ц1 HEPES-CZB medium and centrifuged at 300 x g for 2 min, and the cell pellet was resuspended in 30 ц1 HEPES-CZB medium. The cumulus cell suspension (1 part) was thoroughly mixed with 9 parts 10% PVP in HEPES-CZB containing 0.01% polyvinyl pyrrolidone (PVP) without BSA. Cumulus cells (approximate diameter, 10 to 12 |im) were aspirated gently into and out of an injection pipette (approximate inner diameter, 6 |im) until nuclei were largely devoid of visible cytoplasmic contents. Isolated nuclei were transferred into a new 10% PVP drop and washed; 5 to 8 karyoplasts (extracted nuclei) then were aspirated into the injection pipette for ICNI. Continue reading