Lynn M. Wiley

Epidermal growth factor receptor mRNA and protein increase after the four-cell preimplantation stage in murine development

Epidermal growth factor receptors (EGF-Rs) are expressed at increasing levels on mouse preimplantation embryos. Immunofluorescence assays were used to show that unfertilized eggs and 2-cell embryos have a very low level of reactivity to antimouse EGF-R antibodies, but by the 4-cell stage and later the reactivity increases. The synthesis of EGF-R protein was verified at the blastocyst stage by immunoprecipitation of a 170-kDa metabolically labeled protein. The EGF-R protein is expressed on cell plasma membrane surfaces, but after compaction at the 8-cell stage concentrates on the apical cell surfaces. We also find a low level of expression of EGF-R protein on inner cell mass cells; thus, all cell lineages express receptors from the beginning of gestation. The receptor protein synthesized by the 8-cell embryo and later is probably translated from embryonic transcription, since reverse transcription-polymerase chain reaction indicates increasing levels of mRNA starting after the 4-cell stage. However, we also detected maternal mRNA in zygotes and 2-cell embryos. The pervasive nature of EGF-R expression throughout development suggests important roles for these receptors which could include autocrine and paracrine stimulation.

Cavitation in the mouse preimplantation embryo: NaK-ATPase and the origin of nascent blastocoele fluid☆

This study tested the proposition that Na/K-ATPase activity could be involved in the morphogenetic aspects of mouse blastocyst formation by facilitating the localization of certain organelles to apposed borders, the production of nascent blastocoele fluid, and cavitation. It was assumed that such Na/K-ATPase activity would be sensitive to varying concentrations of external K (Ko)--which would alter plasma membrane potentials--and to ouabain--which would directly alter Na/K-ATPase function. Morulae were cultured for 40 hr in varying concentrations of Ko and/or ouabain and were observed for their ability to form blastocoeles (cavitate) and to localize mitochondria to apposed cell borders. Cavitation was accelerated when Ko was decreased from the control value of 6.0 to 0.6 mM and was delayed when Ko was increased to 25 mM. With Ko at 6.0 mM, 10(-5) M ouabain accelerated cavitation while 10(-4) M ouabain delayed cavitation and reduced the total number of embryos that cavitated by the end of the 40-hr culture period. With Ko at 0.6 mM, 10(-5) M ouabain now delayed cavitation while 10(-4) M ouabain almost completely inhibited it. When Ko was increased to 25 mM, 10(-5) M ouabain again accelerated cavitation while 10(-4) M ouabain delayed-rather than inhibited--cavitation. Morphometric analyses at the electron microscopic level showed changes in the distances of mitochondria from apposed cell borders with conditions that accelerated or delayed cavitation and these changes differed for inside and outside cells of the morula. These observations are consistent with the proposition that Na/K-ATPase activity could be involved in the localization of organelles to apposed cell borders, the production of nascent blastocoele fluid, and in cavitation during mouse blastocyst development.

Gap junction intercellular communication mediates the competitive cell proliferation disadvantage of irradiated mouse preimplantation embryos in aggregation chimeras

Gap junction intercellular communication (GJIC) is thought to play a role in the growth modulation that occurs within cell populations. An example of heterologous growth inhibition (competitive cell proliferation disadvantage) occurs within mouse aggregation chimeras comprised of irradiated and nonirradiated cleavage-stage embryos. The goal of this investigation was to test the hypothesis that GJIC participates in the competitive cell proliferation disadvantage that is expressed by the irradiated embryo in aggregation chimeras. Specifically, we tested the capacity of the GJIC inhibitor 18 alpha-glycyrrhetinic acid (AGA) to inhibit competitive cell proliferation disadvantage in heterologous aggregation chimeras that were comprised of one embryo that was irradiated with 1.0 Gy of (137)Cs gamma rays and then paired with one nonirradiated embryo. We found that AGA successfully inhibited fluorescent dye transfer between irradiated and nonirradiated embryos in heterologous chimeras. Chronic exposure to AGA prevented competitive cell proliferation disadvantage in these radiation chimeras, while exposure to AGA for the first 15 h of culture (prior to gap junction development) did not prevent competitive cell proliferation disadvantage. An unexpected observation was the apparent lack of any effect of inhibiting GJIC by exposure to AGA on blastocyst formation and cell number allocation in the two principal stem cell lineages of the preimplantation mammalian embryo, trophectoderm and inner cell mass.

Impaired cell proliferation in mice that persists across at least two generations after paternal irradiation

Irradiation of male F0 mice 6 to 7 weeks prior to mating causes significant changes in the proliferation of F1 and F2 embryonic cells. These changes are revealed as a competitive cell proliferation disadvantage in chimera assays when the affected embryo is paired with a normal embryo in an aggregation chimera. This effect has been observed previously to be transmitted to F1 embryos for absorbed doses from 0.01 to 1.0 Gy; 0.01 Gy is about 100-fold lower than detectable using conventional germline mutation assays. However, until now there has been no reported cross-generation heritability. We now report that this competitive cell proliferation disadvantage persists without degradation in the F2 generation of embryos when F0 males received 1.0 Gy from gamma irradiation 6 and 7 weeks prior to conception of F1 males.

Antibodies to a renal Na+/glucose cotransport system localize to the apical plasma membrane domain of polar mouse embryo blastomeres

Mouse preimplantation embryos were examined for the cell surface expression of epitopes that cross-react with antibodies to a 75-kDa subunit of a purified porcine renal brush border Na+/glucose cotransport system. A Na+ cotransport system is hypothesized to reside in the apical plasma membrane domain of mouse polar blastomeres and to be associated with the induction of their apical-basal polarity. Western blot analysis showed that unfertilized oocytes as well as preimplantation embryos contain a cross-reacting antigen with an apparent molecular weight of about 75,000. Embryos and their isolated blastomeres were double-labeled and assayed by indirect immunofluorescence (IIF) for the expression of epitopes (visualized by labeling with rabbit antiserum or mouse monoclonal IgG to cotransporter followed by the appropriate rhodamine-conjugated second antibodies) and for the development of cell surface polarity (visualized by the apical restriction of fluoresceinated succinylated concanavalin A binding; FS Con A). IIF did not detect these epitopes until after the second cleavage when 4-cell embryos expressed low-to-moderate levels. Although epitopes were expressed on all surfaces of 4-cell blastomeres, some blastomeres expressed more epitopes on their apical surfaces than on their basolateral ones. All precompaction 8-cell embryos expressed epitopes, with expression being greater apically on some blastomeres. The level of expression appeared to reach a maximum on morulae and to decline on cavitating embryos. Assays performed on isolated blastomeres from postcompaction embryos showed that by the 16-cell stage epitope expression appeared to become restricted to FS Con A-labeled apical plasma membrane domains and was no longer evident on basolateral domains. This apparent apical restriction of epitope expression was confirmed by electron microscopic examination of immunogold-labeled isolated polar 16-cell blastomeres. These results demonstrate that preimplantation mouse embryos contain an antigen(s) that is immunologically and structurally similar to a 75-kDa renal Na+/glucose cotransporter. The onset of cell surface expression of this antigen precedes development of the stable polar phenotype.

The EGFR Gene Family in Embryonic Cell Activities

Publisher Summary This chapter discusses the recent findings on the common threads, connecting the activities of the epidermal growth factor receptor (EGFR) and its ligands in embryonic development, in tissue function and in malignant transformation. The EGFR may be one of the most influential of all the receptor proteins, because it is at the crux of several signaling pathways. It is activated in response to its growth factor ligands in a spatially and temporally appropriate manner during development to stimulate either proliferation, cell death, cell survival, cell locomotion, cell polarization, or differentiation, and it can share some of these responsibilities, if necessary, with its family members. The EGFR is activated in response to the environmental stresses, such as irradiation and hydrogen peroxide, and generates signals that can lead to protective responses. EGFR is involved in activities vital to life, such as stem cell renewal and absorption of nutriments. EGFR can interact, with all its family members, giving graded responses to individual ligands, and hence specificity of responses. The EGFR system does not signal along one pathway. In fact, it is one of the most interactive systems, with far-reaching effects on the cytoskeleton, locomotion, differentiated function, and cell polarity, with the extracellular matrix and secreted factors that further increase its long reach.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accelerates differentiation of murine preimplantation embryos in vitro

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent teratogen in several animal species, especially during the period of organogenesis. The purpose of this study was to test the hypothesis that TCDD has direct effects on the earliest stages of murine embryonic development, namely the preimplantation stages. Three endpoints were measured: 1) embryo cell number, a measure of embryo viability, 2) competitive embryonic cell proliferation utilizing chimeric embryos, another measure of embryo viability, and 3) cavitation rate, a functional measure of trophectoderm differentiation. Neither embryo cell numbers nor competitive embryonic cell proliferation (chimera assay) were affected by TCDD, either when the mother was dosed in vivo (prior to fertilization), or when 2-cell embryos were dosed in vitro. However, cavitation rates of in vitro-dosed embryos, in the presence of 10% fetal bovine serum, were significantly higher for TCDD than controls, suggesting that TCDD accelerated differentiation of murine preimplantation embryos. Taken together, these results demonstrate that: 1) TCDD can act directly on the murine preimplantation embryo, and 2) TCDDs actions are primarily on accelerated differentiation and not on embryo viability. To our knowledge, this is the earliest stage of mammalian development during which TCDD has been shown to exert an effect.

Influence of Short-Term Maternal Zinc Deficiency on the In Vitro Development of Preimplantation Mouse Embryos

Abstract In this study, we evaluated the use of mouse preimplantation embryos as a model to study zinc deficiency-induced abnormal development. In Experiment 1, the effect of culture medium Zn concentrations on blastocyst development was studied. Preimplantation embryos (2 and 4 cells) obtained from superovulated females developed normally in media containing 0.7–30 μM Zn for up to 72 hr; higher levels of medium Zn resulted in abnormal development. In Experiment 2A, females were fed diets containing 50 (+Zn) or 0.4 (-Zn) μg Zn/g (760 vs 6 nmol/g, respectively) from 1 day before to 1 day after mating (3 days total). Preimplantation embryos were removed from the dams and cultured for 72 hr in 0.7 μM Zn medium. Embryos from the –Zn dams were morphologically normal at time zero; however, over the 72-hr period, these embryos tended to develop at a slower rate than controls, although compaction and cavitation frequency were similar. By the end of the 72-hr culture period, embryos from –Zn dams had significantly fewer cells than did embryos from control dams. In Experiment 2B, an extended period of maternal Zn deprivation (6 days) was used to investigate the potential for further impairment of in vitro preimplantation embryo development observed in Experiment 2A. Results from this experiment were consistent with those from Experiment 2A, in addition to providing evidence that the developmental progress of embryos obtained from mice fed Zn-deficient diets for 6 days was significantly impaired. In Experiment 3, the potential for supplemental Zn in culture medium to overcome the impairment in development due to maternal Zn deficiency was investigated. Embryos from female mice subjected to the same dietary regimen described in Experiment 2A were cultured to the blastocyst stage in medium containing Zn at a concentration of either 0.7 or 7.7 μM. Medium Zn supplementation did not improve development of embryos from dams fed Zn-deficient diets. In summary, embryos from mice fed –Zn diets for a 3-or 6-day period encompassing oocyte maturation and fertilization exhibited impaired development in vitro. This impairment was not overcome by medium Zn supplementation.

Influence of antioxidants on cadmium toxicity of mouse preimplantation embryos in vitro

To test the hypothesis that the developmental toxicity of cadmium (Cd) is due in part to oxidative damage, embryos were cultured in medium containing 0.0, 1.0, 3.0, or 6.0 microM Cd with or without various antioxidants for 72 h. Ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and glutathione (GSH) were all effective at ameliorating 1.0 microM Cd-induced embryotoxicity. For embryos cultured in medium containing either 3.0 or 6.0 microM Cd, GSH was effective at ameliorating Cd toxicity while the other antioxidants tested were ineffective. Pretreating embryos with antioxidants for 24 h prior to exposing them to Cd and antioxidants did not significantly alter the previously observed improvement with the exception that pretreatment with GSH virtually eliminated Cd-induced embryotoxicity between 1.0 and 6.0 microM Cd. A 4-h exposure to GSH prior to culture in Cd markedly improved embryo development suggesting that GSH taken up during pretreatment can provide protection against Cd-induced embryotoxicity. This work supports the hypothesis that the developmental toxicity of Cd is in part due to oxidative damage that can be modulated by select antioxidants.

Expression and function of amphiregulin during murine preimplantation development

Amphiregulin (Ar) is an EGF receptor ligand that functions to modulate the growth of both normal and malignant epithelial cells. We asked whether mouse preimplantation embryos express Ar, and if so, what the function of Ar is during preimplantation development. We used RT‐PCR to show expression of Ar mRNA in mouse blastocysts, and using a polyclonal anti‐Ar antibody and indirect immunofluorescence, we detected the presence of Ar protein in morula‐ and blastocyst‐stage embryos. Ar protein was present in both the cytoplasm and nucleus in both morulae‐ and blastocyst‐stage embryos, which is similar to Ar distribution in other cell types. Embryos cultured in Ar developed into blastocysts more quickly and also exhibited increased cell numbers compared to control embryos. In addition, 4‐cell stage embryos cultured in an antisense Ar phosphorothioate‐modified oligodeoxynucleotide (S‐oligo) for 48 hr exhibited slower rates of blastocyst formation and reduced embryo cell numbers compared to embryos exposed to a random control S‐oligo. TGF‐α significantly improved blastocyst formation, but not cell numbers, for embryos cultured in the antisense Ar S‐oligo. From these observations, we propose that Ar may function as an autocrine growth factor for mouse preimplantation embryos by promoting blastocyst formation and embryo cell number. We also propose that blastocyst formation is stimulated by Ar and TGF‐α, while Ar appears to exert a greater stimulatory effect on cell proliferation than does TGF‐α in these embryos. Mol. Reprod. Dev. 47:271–283, 1997.