Effects of growth factors FGF4, TGFα, and TGFβ1 on development of parthenogenetic embryos of C57BL/6 mice

We studied the effects of three growth factors, fibroblast growth factor (FGF4), transforming growth factor (TGF), and transforming growth factor 1 (TGF1), on development of diploid parthenogenetic embryos of C57BL/6 mice, which are not capable of developing to somatic stages. Parthenogenetic embryos were treated with growth factors at optimal doses in vitro at the morula-blastocyst stages and transplanted in the uterus of pseudopregnant females. FGF4 and TGF improved the development of parthenogenetic embryos at the preimplantation stages and the number of blastocysts increased under the influence of TGF. All three growth factors improved the implantation of embryos in the uterus. When FGF4 or TGF1 2.4 were added to the nutrient medium, 2.4 or 1.6%, respectively, of parthenogenetic embryos reached the somatic stages in utero. No somitic embryos were observed in the control. The treatment of parthenogenetic embryos with two growth factors, FGF4 and TGF1 , simultaneously increased the amount of somatic embryos to 7.5%, while combination of three growth factors in creased the amount of such embryos to 16.7%. In the latter case, some parthenogenetic embryos reached the stage of 25–27 pairs of somites and were 2.0–2.5 mm long. The data we obtained suggest that, when combined, the growth factors FGF4, TGF, and TGF1 possessed a synergistic effect leading to a significant improvement of the development of parthenogenetic C57BL/6 embryos.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 145–150.Original Russian Text Copyright © 2005 by Penkov, Platonov, Dimitrov, Mironova, Konyukhov.     

The effect of fibroblast growth factors (FGF-2, FGF-4) on the development of parthenogenetic mouse embryos

We studied the effects of two growth factors, FGF-2 and FGF-4, on development of diploid parthenogenetic mouse embryos (CBA x C57BL/6)F1. Parthenogenetic embryos were treated with FGF-2 or FGF-4 in vitro at the morula stage and, after they reached the blastocyst stage, transplanted into the uteri of pseudopregnant females. FGF-2 and FGF-4 did not affect the number of blastocysts formed in vitro or implantation into the uterus. However, FGF-2 and FGF-4 at optimal doses decreased the mortality rate of parthenogenetic embryos at the early postimplantation stages and increased twofold the number of embryos that developed in utero to the somite stages: 42 and 36%, respectively, versus 20% in the control. The results obtained suggest that the treatment of parthenogenetic mouse embryos with FGF-2 or FGF-4 modulate the effects of genomic imprinting and prolong the development of parthenogenetic embryos at the postimplantation stages.     

Transforming growth factor alpha (TGFalpha) modulates the effect of genomic imprinting and prolongs the development of parthenogenetic murine embryos]

The effect of transforming growth factor alpha (TGF alpha) on the development of diploid parthenogenetic mouse embryos (CBA x C57BL/6)F1 was studied. The embryos were in vitro treated with the TGF alpha at the stage of morula. Upon reaching the blastocyst stage, each embryo was implanted into uterus of a pseudopregnant female. At a dose of 5 ng/ml, the TGF alpha was found to improve development of parthenogenetic embryos before implantation, increase significantly the number of developing blastocysts, and promote embryo implantation into uterus. After treatment with TGF alpha at a dose of 10 ng/ml, 4% of parthenogenetic embryos reached the stage of 30-45 somites and had forelimb and hindlimb buds; the embryo size from vertex to sacrum was 2.0 to 3.8 mm. A well-developed placenta was observed in 6% of TGF alpha-treated parthenogenetic embryos that reached the somite stages. In the parthenogenetic embryos with the most prominent development (42-45 somites) treated with 10 ng/ml of TGF alpha, the placental diameter was 4.0 to 4.2 mm on day 12 of gestation, which is close to the placental size of the normal (fertilized) 11-day-old mouse embryos. Our results suggest that endogenous TGF alpha can modulate the effects of genomic imprinting significantly improving formation of trophoblast derivatives and promoting longer postimplantation development of parthenogenetic embryos.     

Leukemia inhibitory factor (LIF) improves and prolongs the development of parthenogenetic mouse embryos

We studied the effect of the growth factor LIF on the development of parthenogenetic mouse embryos (CBA x C57BL/6)F1. LIF was added to the culture medium at 10, 50, 100, and 250 ng/ml at the morula stage and parthenogenetic embryos were cultivated in vitro until the late blastocysts stage and then transplanted in the uterus of pseudopregnant females, which were then sacrificed on day 12 of pregnancy. All the LIF doses used improved the development of parthenogenetic mouse embryos at the preimplantation stages and increased the amount of blastocysts by 16%, on average, as compared to the control. LIF at 50 and 100 ng/ml increased approximately twice the number of embryos that reached the somatic stages. Some of them reached the stage of 32-45 somites and had fore and hind limb buds. No such embryos were found in the control. Well formed placenta was observed in 6% of the embryos treated with LIF and the most pronounced effect was recorded at 100 ng/ml. The data we obtained suggest that exogenous LIF can improve pre- and postimplantation development of parthenogenetic mouse embryos due, possibly, to increased survival rate of embryonic stem cells derived from the inner cell mass of blastocysts. LIF improves not only the development of the parthenogenetic embryo per se, but also the formation of its extraembryonic envelopes, which leads to the development of a larger placenta in LIF-treated parthenogenetic embryos, as compared to the control.     

Effects of growth factors FGF2 and IGF2 on the development of parthenogenetic mouse embryos in utero and in vitro

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA x C57BK/6)F1. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18-21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 micrograms/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryos in vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatment in vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.     

TGFbeta and TGFalpha, antagonistic effect in vitro on extracellular matrix accumulation by chick skin fibroblasts at two distinct embryonic stages

ECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor beta (TGFbeta) and alpha (TGFalpha) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFbeta on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFalpha and TGFbeta production, in particular TGFbeta3 and TGFbeta4, and the number of TGFbeta receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFbeta. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFbeta has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFbeta3 and TGFbeta4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFbeta3 and TGFbeta4 mRNA expression is increased in the first stages of development. Instead, the level of TGFalpha increases in successive developmental stages. Since TGFalpha stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFbeta, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFbeta effect. Overall our data suggest that the production of TGFbeta and TGFalpha are age-dependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation.     

Leukemia Inhibitory Factor (LIF) Improves and Prolongs the Development of Parthenogenetic Mouse Embryos

We studied the effect of the growth factor LIF on the development of parthenogenetic mouse embryos (CBA × C57BL/6)F1. LIF was added to the culture medium at 10, 50, 100, and 250 ng/ml at the morula stage and parthenogenetic embryos were cultured in vitro until the late blastocyst stage and then transplanted in the uterus of pseudopregnant females, which were then sacrificed on day 12 of pregnancy. All the LIF doses used improved the development of parthenogenetic mouse embryos at the preimplantation stages and increased the amount of blastocysts by 15%, on average, as compared to the control. LIF at 50 and 100 ng/ml increased approximately twice the number of embryos that reached the somite stages. Some of them reached the stage of 32–45 somites and had fore and hind limb buds. No such embryos were found in the control. Well formed placenta was observed in 6% of the embryos treated with LIF and the most pronounced effect was recorded at 100 ng/ml. The data we obtained suggest that exogenous LIF can improve pre- and postimplantation development of parthenogenetic mouse embryos due, possibly, to increased survival rate of embryonic stem cells derived from the inner cell mass of blastocysts. LIF improves not only the development of the parthenogenetic embryoper se, but also the formation of its extraembryonic envelopes, which leads to the development of a larger placenta in LIF-treated parthenogenetic embryos, as compared to the control.     

Effects of Growth Factors FGF2 and IGF2 on the Development of Parthenogenetic Mouse Embryos in utero and in vitro

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA × C57BK/6)FF₁. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18–21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 g/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). More than a half of FGF-2-treated parthenogenetic embryos developed to the stage of 40 and some of them, to the stage of 50 somites. The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryosin vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatmentin vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.     

Expression of imprinted Igf2 and Peg1/Mest genes in postimplantation parthenogenetic mouse embryos treated with transforming growth factor alpha in vitro

The effect of transforming growth factor alpha (TGFt) on the expression of imprinted Igf2 and Peg1/Mest genes was studied in diploid parthenogenetic embryos (PEs) of (CBA x C57BL/6)F1 mice during the postimplantation period of embryogenesis. The PEs were treated with TGFalpha in vitro at the morula stage and, after they developed to the blastocyst stage, were implanted into the uterus of false-pregnant females. On the tenth day of pregnancy, the PEs were explanted for subsequent in vitro culturing for 24 or 48 h. The expression of the imprinted Igf2 and Peg1/Mest genes was studied by means of whole mount in situ hybridization using digoxigenin-labeled antisense RNAs. The expression of the imprinted Igf2 and Peg1/Mest genes was studied in embryos on the tenth day of in utero development before culturing and after 24 and 48 h of culturing in vitro. The expression of Igf2 before culturing was detected only in the brain of 60% of PEs on the tents day of pregnancy (the 21-to 25-somite stages); while the Peg1/Mest expression was not detected at all. In control (not treated with TGFalpha) PEs, neither gene was expressed at the same 21- to 25-somite stages. After 24 h of culturing, the Igf2 expression was detected in the brain of 71% of PEs at the 30- to 35-somite stages, while the Peg1/Mest expression was not detected. In control (untreated) PEs, neither imprinted gene was expressed at the 30- to 35-somite stage. After 48 h of culturing, Igf2 was expressed in the regions of the brain, developing jaws, heart, liver, and somites of all TGFalpha-treated PEs at the 40- to 45-somite stages; and Peg1/Mest was expressed in the brain, heart, and liver of these embryos. In control (untreated) PEs, neither Igf2 nor Peg1/Mest was expressed at these stages The expression patterns of the imprinted Igf2 and Peg1/Mest genes in PEs at the most advanced developmental stages (40-45 somites) and in normal (fertilized) embryos at the same stages were similar; however, their expression rate in PEs was substantially lower than in normal embryos. These data indicate that exogenous TGFalpha can reactivate the expression of the two imprinted genes, modulating the effects of genomic imprinting in such a way that the PE development is improved and substantially prolonged.     

The development of diploid parthenogenetic mouse embryos of the inbred C57BL and CBA strains]

We studied preimplantation development in vitro and postimplantation development in vivo of diploid parthenogenetic mouse embryos of C57BL/6 and CBA strains, as well as of (CBA x C57BL/6)F1 hybrids. Development to blastocyst stage of diploid eggs obtained from C57BL/6, CBA, and hybrid mice was observed in 90, 15, and 73% cases, respectively. After implantation, C57BL/6 embryos did not develop to somite stages, while CBA and hybrid embryos reached various stages of somite formation in 45 and 30% cases, respectively. Cultivation of embryos beginning from one-cell stage in the medium containing 2% newborn calf serum increased the yield of blastocysts from 15 to 59% in CBA embryos and from 73 to 90% in hybrids; However, such effect was not observed with C57BL/6 embryos. The latest stages of development observed in CBA and hybrid diploid parthenogenetic embryos were 33-35 somites and 25-30 somites, respectively. Imprinting patterns in chromosomes of CBA and C57BL/6 gametes are discussed.     

TGFalpha reactivates imprinted Igf2 in the parthenogenetic mice embryos and placenta

Imprinted genes play important roles in the mammalian development. In the parthenogenetic embryos (PE) there is only expression of maternally expressed genes. Therefore, PEs are appropriate experimental models to study genomic imprinting controlling mechanisms. The maternally expressed H19 and paternally expressed Igf2 are reciprocally imprinted genes in normal embryos. Here we studied effect of transforming growth factor alpha (TGFalpha) treatment in vitro (10 ng/ml at the morula stage) on the expression of Igf2/H19 locus in mice PE (9.5-days of gestation, 25 somites) and their placentas (PP). Using RT-PCR we showed that TGFalpha reactivated maternally imprinted Igf2 gene in parthenogenetic embryos and placentas. In spite of similar Tgfalpha expression in the pre-implantation stages, its expression in the 9.5-day parthenogenetic embryos is significantly less than in normal embryos (NE). In our experiments it was shown that reactivation of Igf2 gene occurred independently of H19 gene. In vitro TGFalpha treatment of mouse PE reactivated paternally expressed Igf2 gene in the PE and PP. In the PE and PP both Igf2 and H19 were expressed. It seems that TGFalpha can play an important role as modulator of the Igf2/H19 locus.     

Transforming Growth Factor α (TGFα) Modulates the Effects of Genomic Imprinting and Prolongs Development of Parthenogenetic Mouse Embryos

The effect of transforming growth factor (TGF) on the development of diploid parthenogenetic mouse embryos (CBA × C57BL/6)F₁was studied. The embryos were in vitro treated with the TGF at the morula stage. Upon reaching the blastocyst stage, each embryo was implanted into uterus of a pseudopregnant female. At a dose of 5 ng/ml, the TGF was found to improve development of parthenogenetic embryos before implantation, increase significantly the number of developing blastocysts, and promote embryo implantation into uterus. After treatment with TGF at a dose of 10 ng/ml, 4% of parthenogenetic embryos reached the stage of 30–45 somites and had forelimb and hindlimb buds; the crown rump length of the embryo size from vertex to sacrum was 2.0 to 3.8 mm. A well-developed placenta was observed in 6% of TGF-treated parthenogenetic embryos that reached the somite stages. In the parthenogenetic embryos with the most prominent development (40–45 somites) treated with 10 ng/ml of TGF, the placental diameter was 4.0 to 4.2 mm on day 12 of gestation, which is close to the placental size of the normal (fertilized) 11-day-old mouse embryos. Our results suggest that exogenous TGF can modulate the effects of genomic imprinting significantly improving formation of trophoblast derivatives and promoting longer postimplantation development of parthenogenetic embryos.     

Opposing actions of TGFbeta and MAP kinase signaling in undifferentiated hen granulosa cells

The present studies were conducted to establish interactions between transforming growth factor (TGF)-beta and the epidermal growth factor (EGF) family members, TGFalpha and betacellulin (BTC), relative to proliferation and differentiation of granulosa cells in hen ovarian follicles. Results presented demonstrate expression of TGFbeta isoforms, plus TGFalpha, BTC, and ErbB receptors in prehierarchal follicles, thus establishing the potential for autocrine/paracrine signaling and cross-talk within granulosa cells at the onset of differentiation. Treatment with TGFalpha or BTC increases levels of TGFbeta1 mRNA in undifferentiated granulosa cells, while the selective inhibitor of mitogen activated protein kinase signaling, U0126, reverses these effects. Moreover, TGFbeta1 attenuates c-myc mRNA expression and granulosa cell proliferation, while TGFalpha blocks both these inhibitory effects. Collectively, these data provide evidence that EGF family ligands regulate both the expression and biological actions of TGFbeta1 in hen granulosa cells, and indicate that the timely interaction of these opposing factors is an important modulator of both granulosa cell proliferation and differentiation.     

Effects of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), and 2,3,7,8-tetrachlorodibenzo-p-dioxin on fusion of embryonic palates in serum-free organ culture using wild-type, EGF knockout, and TGFalpha knockout mouse strains

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing cleft palate (CP). TCDD exposure disrupts expression of epidermal growth factor (EGF) receptor, EGF, and transforming growth factor-alpha (TGFalpha) in the palate and affects proliferation and differentiation of medial epithelial cells. EGF knockout embryos are less susceptible to the induction of CP by TCDD. This study used palate organ culture to examine the hypothesis that EGF enables a response to TCDD. METHODS: The midfacial tissues from wild-type (WT), EGF knockout, C57BL/6J, and TGFalpha knockout embryos were placed in organ culture on gestational day (GD) 12. Palatal explants were cultured for 4 days in serum-free Bigger's (BGJ) medium with 0.1% dimethyl sulfoxide (DMSO) or 1 x 10(-8) M TCDD with or without 2 ng of EGF/ml, 1 or 2 ng of TGFalpha/ml. Effects on palatal fusion were evaluated on day 4 of culture. EGF levels in explants and medium were determined using Luminex technology. RESULTS: In serum-free, control medium, palates from all of the strains fused. EGF knockout palates cultured with TCDD (no EGF) fused, but those cultured with TCDD + 2 ng of EGF/ml failed to fuse (p < 0.05 vs. control or TCDD without EGF). TGFalpha knockout palates failed to fuse when cultured with TCDD + 2 ng of TGFalpha/ml. EGF levels increased in tissue and accumulated in the medium after 24 hr of culture. CONCLUSIONS: This study demonstrated that providing EGF to the palates of EGF knockout mice restored the response to TCDD. These studies support the hypothesis that the mechanism for induction of CP by TCDD is mediated via the EGFR pathway.     

Heparin binding epidermal growth factor-like growth factor is a transforming growth factor beta-regulated gene in intestinal epithelial cells.

Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an EGF-related peptide with prominent effects on cell growth and migration. We explored potentially unique characteristics of HB-EGF in the intestinal epithelial cell line RIE-1. HB-EGF stimulated [(3)H]thymidine incorporation to a level equivalent to transforming growth factor alpha (TGFalpha). HB-EGF also rapidly activated MAPK and induced cyclin D1 in mid-G1 with kinetics similar to TGFalpha. Unlike TGFalpha, HB-EGF mRNA was induced within 1 h by a variety of stimuli, including TGFbeta1. Maximal induction by TGFbeta (7-fold) occurred within 2 h of treatment. Actinomycin D decay curves showed that TGFbeta1 had no effect on HB-EGF mRNA half-life (T(1/2) 20 min). Induction of HB-EGF by TGFbeta1 was not affected by pretreatment with the MEK inhibitor PD-98059 while inhibition of protein kinase C either partially (calphostin C) or completely (staurosporin) blocked induction. Our results suggest that major differences exist in the regulation of the closely related EGF family members TGFalpha and HB-EGF. TGFbeta and HB-EGF, structurally unrelated peptides with potent effects on wound healing, may function coordinately to mediate responses to wounding or cell injury in the intestinal epithelium.     

Different influences of genomic imprinting on the development of parthenogenetic cell clones in C57BL/6 and CBA mice

Clonal analysis of parthenogenetic chimeric mouse embryos C57B1/6(PG)<-->BALB/c has shown that parthenogenetic cell clones C57BL/6 are present in the brain, liver, and kidneys of 14- and 18-day-old embryos. The content of the parthenogenetic component (PG) in these organs on day 18 was lower than on day 14, and, in some 18-day-old embryos, parthenogenetic cell clones were absent from the liver and/or kidneys. These data suggest that, during the embryogenesis of parthenogenetic chimeras, parthenogenetic cell clones of mostly endodermal and mesodermal origins were actively eliminated. Therefore, in such parthenogenetic adult chimeras, parthenogenetic clones of mostly ectodermal origins were preserved. In parthenogenetic chimeras CBA(PG)<-->BALB/c, parthenogenetic cell clones were actively eliminated at early embryonic stages, and, as a result, they were absent at the post-implantation stages. Hence, during development of parthenogenetic cell clones, the effects of genomic imprinting are expressed unequally in C57BL/6 and CBA mice.     

Teratogenic effects of retinoic acid are modulated in mice lacking expression of epidermal growth factor and transforming growth factor-alpha

BACKGROUND: Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) regulate cell proliferation and differentiation in the embryo. The induction of cleft palate (CP) by all trans-retinoic acid (RA) was associated with altered expression of TGFalpha, EGF receptor, and binding of EGF. This study uses knockout (KO) mice to examine the roles of EGF and TGFalpha in teratogenic responses of embryos exposed to RA. METHODS: Pregnant wild-type (WT) mice of mixed genetic background, EGF KO, C57BL/6J, and TGFalpha KO mice were given a single oral dose of RA (100 mg/kg, 10 ml/kg) or corn oil on GD 10 at 12 PM, GD 11 at 12 PM or 4 PM, or GD 12 at 8 AM or 12 PM (plug day = GD 0). GD 18 fetuses were examined for external, visceral, and skeletal effects. RESULTS: After exposure to RA on GD 12, the incidence of CP in EGF KO was significantly reduced relative to WT. In TGFalpha KO fetuses, RA exposure on GD 10 increased the incidence of CP versus C57BL/6J. The incidence of skeletal defects in the limbs, vertebrae, sternebrae, and ribs were also affected by lack of expression of EGF or TGFalpha with region-specific amelioration or exacerbation of the effects of RA. In TGFalpha KO fetuses, incidences of forelimb long bone and digit defects increased relative to C57BL/6J. In EGF KO fetuses, relative to WT, the incidence of hindlimb oligodactyly was increased. In EGF KO, but not WT, RA produced short, bent radius, humerus, and ulna. Both TGFalpha and EGF KO mice had increased incidences of dilation of the renal pelvis and this was reduced by RA. CONCLUSIONS: RA exposure produced skeletal and visceral defects in all genotypes; however, EGF or TGFalpha KO influenced the incidence and severity of defects. This study supports a role for EGF and TGFalpha in the response to RA.     

Development of mouse embryos after immunoneutralization of mitogenic growth factors mimics that of cloned embryos

The extent to which mitogenic growth factors influence embryo development is not well characterized. We sought to determine the effect of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) on naturally fertilized (in vivo-derived) and in vitro-fertilized mouse embryos, compared with that on cloned (intracytoplasmic nuclear injection-derived) mouse embryos, in which EGF and TGFalpha expression is markedly reduced. Immunoneutralization of EGF, TGFalpha, and EGF receptor by using specific antibodies significantly reduced the blastocyst development rate (in vivo-derived: 66%, 63%, and 63%, respectively; in vitro-fertilized: 57%, 55%, and 56%, respectively), increased the number of apoptotic nuclei (in vivo-derived: 9%, 10%, and 9%, respectively; in vitro-fertilized: 13%, 13%, and 13%, respectively), decreased the total number of cells (in vivo-derived: 87%, 85%, and 86%, respectively; in vitro-fertilized: 86%, 85%, and 86%, respectively), and increased the inner cell mass:trophectoderm ratios (in vivo-derived: 1:2.70 +/- 0.05, 1:2.73 +/- 0.04, 1:2.71 +/- 0.06, respectively; in vitro-fertilized: 1:2.94 +/- 0.02, 1:2.96 +/- 0.02, 1:2.95 +/- 0.02, respectively). In most cases, combined treatment with neutralizing antibodies to both EGF and TGFalpha accentuated changes in these parameters. Further, the effect of combined immunoneutralization on these parameters in fertilized embryos was no different from those in cloned embryos. Therefore, normal expression of mitogenic growth factors is crucial for successful development of mouse embryos before implantation. Inhibiting the action of mitogenic growth factors causes fertilized embryos to exhibit developmental characteristics similar to those of cloned embryos, which may partially explain the poor developmental potential of cloned mammalian embryos.     

Different Influences of Genomic Imprinting on the Development of Parthenogenetic Cell Clones in C57BL/6 and CBA Mice

Clonal analysis of parthenogenetic chimeric mouse embryos C57BL/6(PG) BALB/c has shown that parthenogenetic cell clones C57BL/6 are present in the brain, liver, and kidneys of 14- and 18-day-old embryos. The content of the parthenogenetic component (PG) in these organs on day 18 was lower than on day 14, and, in some 18-day-old embryos, parthenogenetic cell clones were absent from the liver and/or kidneys. These data suggest that, during the embryogenesis of parthenogenetic chimeras, parthenogenetic cell clones of mostly endodermal and mesodermal origins were actively eliminated. Therefore, in such parthenogenetic adult chimeras, parthenogenetic clones of mostly ectodermal origins were preserved. In parthenogenetic chimeras CBA(PG) BALB/c, parthenogenetic cell clones were actively eliminated at early embryonic stages, and, as a result, they were absent at the post-implantation stages. Hence, during development of parthenogenetic cell clones, the effects of genomic imprinting are expressed unequally in C57BL/6 and CBA mice.     

Effect of 5-azadeoxycytidine and retinoic acid on expression of genomic imprinting in parthenogenetic mouse embryos

The action of two types of substances has been studied: 5-azadeoxycytidine and retinoic acid, which have a demethylation effect on DNA in the development process of diploid parthenogenetic mouse embryos. The effect of 5-azadeoxycytidine on hybrid mice (CBA × C57BL/6)F1 in vitro for 6 h, in the presence of single cell parthenogenetic embryos during the S-phase of the cell cycle has been studied. After developing to the blastocyst stage in vitro, parthenogenetic embryos were transplanted into the uterus of false pregnant females. It has been determined that a concentration of 0.1 μM 5-azadeoxycytidine activates embryonic development in the preimplantation period until the blastocyst stage (69% in experiment; 61% in the control) and during postimplantation, it increases the number of available space in the uterus for implantation (76% in experiment; 63% in the control).