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Formation of fetal rat cardiac cell clones by retroviral transformation: retention of select myocyte characteristics.

Ventricular myocyte (cardiomyocyte) growth is exquisitely regulated such that embryonic and fetal development are the primary periods of active cellular division. This report describes formation of three separate cardiomyocyte cell clones obtained by replication-defective retroviral (v/myc and v-H-ras) transformation of primary cultures of day-16 fetal rat cardiomyocytes. The cell clones do not spontaneously contract, yet they express several cardiac-specific (cardiac troponin-C, alpha-cardiac actin) and associated genes (Connexin 43, Early growth response gene-1) with stable expression of several genes determined through the 28th passage. None of these cell clones express skeletal muscle actin or the skeletal muscle regulatory gene MyoD1; yet all display ultrastructural and biochemical evidence of their cardiac muscle lineage. Molecular and biochemical studies of cardiac-specific gene regulation can be anticipated from the cell clones as it pertains to nuclear transcription factors and transient CAT-based reporter gene constructs. The formation of these cell clones will enable further studies of growth and development of this unique muscle cell population of the cardiovascular system to be performed at the cellular and molecular level.

Animals↗

Proteolysis and developmental signal transduction.

Regulated proteolysis is a critical feature of many intercellular signalling pathways that control cell-fate specification and tissue patterning during metazoan development. The roles of proteolysis in three different pathways, the Toll, Hedgehog, and Notch pathways, are described to illustrate the importance of specific protein cleavages in both extracellular ligand-receptor interactions and intracellular signal transduction. An emerging principle is the use of proteolysis to control the maturation and activation of receptors, to limit the spatial diffusion of their ligands, and to modulate the subcellular localization or transcriptional activity of DNA-binding factors in response to receptor-ligand interactions at the cell surface.

Animals↗

H-2 haplotype and the embryotoxicity of serum from nonpregnant congenic mice.

Previous studies revealed a significant association between genes at or near the H-2 complex and fetal loss. Reasoning that the maternal serum might contain one or more unknown factors that are harmful to early embryonic or fetal development, or both, we performed an embryotoxicity screen using chick embryos and serum from nonpregnant C57BL/10Sn (H-2b) and B10.A/SnSg (H-2a) congenic mice. Serum from the strain with the higher frequency of fetal loss (C57BL/10 Sn) yielded a significantly greater frequency of chick abnormality, specifically neural tube malformation and death, than the serum from the strain with the lower frequency of fetal loss (B10.A/SnSg). Further, the C57BL/10 Sn serum demonstrated a highly significant dose-response. These results suggest that analogous studies may be profitable with women who have a history of chronic fetal wastage and/or offspring with neural tube defects.

Animals↗

Effects of sera from patients given various anesthetics on preimplantation mouse embryo development in vitro.

This study compared the effects of sera from patients given various anesthetics on in vitro mouse preimplantation embryo development. Patients electing bilateral laparoscopic tubal sterilization were subjected to general anesthesia with nitrous oxide (N2O) that included either isofluorane (ISO/N2O) as an inhalant or fentanyl or morphine (FEN/MOR/N2O). The addition of sera collected 1 hr after anesthetic induction significantly reduced the numbers of two-cell mouse embryos that developed to blastocyst in the ISO/N2O group as compared to that of preanesthesia sera. In contrast, no detrimental effects were revealed from sera of patients given FEN/MOR/N2O. Comparison of sera from patients given ISO/N2O and FEN/MOR/N2O for laparoscopic oocyte retrieval and from patients given spinal anesthesia and/or i.v. sedation for ultrasonic retrieval also revealed a decrease in mouse embryo development in the ISO/N2O group, but no differences were seen in the other anesthetic regimens. ISO/N2O anesthesia was also associated with a significantly decreased fertilization rate of mature oocytes retrieved. However, no significant effect of ISO/N2O anesthesia on IVF pregnancy rates could be demonstrated. These studies indicate that embryo toxic effects can be detected in sera from patients given ISO/N2O and that this anesthetic may be detrimental to the success of in vitro fertilization and embryo transfer.

Adult↗

Synergy between tumor necrosis factor and endotoxin decreases early embryo development in vitro.

The purpose of the present study was to investigate the individual and combined effects of low levels of endotoxin and physiological levels of tumor necrosis factor on in vitro fertilization and preimplantation embryo development. B6D2F1 mice were superovulated by utilizing pregnant mare serum gonadotropin and human chorionic gonadotropin. Oocyte-cumulus complexes (3221 oocytes) were collected, pooled, and randomized into control and treatment groups. Sperm were collected from the caudae epididymides of mature male mice and allowed to capacitate. Treatments included culture media supplemented with increasing amounts of endotoxin (0.5, 1.0, and 2.0 ng/ml; sp act, 3000 IU/ng) and/or tumor necrosis factor (1, 10, and 50 pg/ml; sp act, 0.01 IU/pg) throughout the fertilization and preimplantation development process. Percentage cleavage and percentage expanded blastocyst formation were evaluated. No significant effects were observed for percentage cleavage or percentage expanded blastocyst formation in either the endotoxin (E) or the tumor necrosis factor (TNF) groups. The combination of endotoxin and tumor necrosis factor at any of the levels tested did not significantly decrease cleavage; however, percentage blastocyst formation was decreased with any combination of TNF and E (P less than 0.05-P less than 0.001). We conclude that TNF and E exert significant synergistic effects which are detrimental to in vitro preimplantation embryonic development.

Animals↗

Preimplantation development following in vitro fertilization of mouse oocytes: effects of timing of superovulation and preincubation in vitro.

The early embryonic development of in vitro fertilized oocytes was assessed following superovulation in F1 hybrid (C57BL/6 X CBA/Ca) mice. Decreasing the time interval between the administration of constant doses of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) resulted in decreases in the frequency of development to the blastocyst stage but had no significant effect on development to the two-cell stage. Preincubation of postovulatory oocytes in vitro prior to insemination did not compensate for the reduced preovulatory development in vivo but resulted in decreases in the frequency of development to the blastocyst stage. The results indicate that inadequate preovulatory development of superovulated mouse oocytes can adversely affect the preimplantation development of in vitro fertilized embryos in the absence of a visible inhibitory effect on development to the two-cell stage and also that preincubation of postovulatory oocytes in vitro prior to fertilization reduces subsequent developmental capacity.

Animals↗

Effects of various degrees of undernutrition of mice on pregnancy and conceptus.

Maternal intake of drastically (1/2 of control) and substantially (1/2 of control) reduced feeding was studied in pregnant mice during gestation and embryonic development. Resorptions, fetal mortality rate and fetal weight were significantly affected by maternal undernutrition during the treatment period. The number of fetuses with intrauterine weight retardation was also significant.

Animals↗

Effect of anordrin on the development of mouse preimplantation embryos in vitro.

PURPOSE: The in vitro effect of anordrin and anordiol on the development of mouse two-cell embryos was studied. METHOD: Female mice were primed with gonadotropins for superovulation and caged with male mice. Preimplantation embryos, at the two-cell stage, were recovered from the oviducts at 40 hr post-hCG. In the first experiment, two-cell embryos were exposed to culture medium containing different concentrations of anordrin for 3, 12, 24, and 80 hr and then grown in the anordrin-free culture medium and assessed for the formation of total and hatching blastocysts at 80 hr. In the second experiment, two-cell embryos were grown in culture medium containing different concentrations of anordiol and assessed for the formation of total and hatching blastocysts at 80 hr in vitro. RESULTS: Exposure of two-cell embryos to anordrin concentrations of 2.5-7.5 micrograms/ml for 12 hr, 2.5-5.0 micrograms/ml for 24 hr, and 2.5 micrograms/ml for 80 hr caused significant inhibition of the formation of total blastocysts and to 2.5-7.5 micrograms/ml for 12 hr, 1.0-2.5 micrograms/ml for 24 hr, and 1.0 micrograms/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts, in a exposure time-dependent and dose-dependent manner. Exposure of two-cell embryos to anordiol concentrations of 15-25 micrograms/ml for 80 hr caused significant inhibition of the formation of total blastocysts and to 15-20 micrograms/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts in a dose-dependent manner. CONCLUSION: Anordrin and its metabolite anordiol inhibit the development of two-cell embryos in vitro.

Animals↗

Anti-platelet activating factor (PAF) antibody inhibits CFW mouse preimplantation embryo development.

OBJECTIVE: Our purpose was to investigate the effect of anti-PAF antibodies on CFW mouse embryo development in vitro. DESIGN: We studied the in vitro development of CFW mouse one-cell-stage embryos cultured in MEM supplemented with anti-PAF, anti-IgG, or MEM alone to the hatched blastocyst stage. RESULTS: Mouse embryos cultured with anti-PAF (1:5 dilution; 61%) significantly decreased embryo development compared to controls (MEM alone; 93%), whereas embryos cultured in anti-mouse IgG-supplemented MEM (1:10 dilution; 93%) had no effect. CONCLUSIONS: The results provide additional evidence that PAF is produced and secreted by cleavage-stage embryos and is required during the preimplantation period.

Animals↗

Preimplantation embryo development enhanced by epidermal growth factor.

PURPOSE: Enhanced embryo maturity and advanced stages of cleavage at the time of embryo transfer are associated with superior pregnancy rates in in vitro fertilization procedures. This study was performed to assess the potential usefulness of epidermal growth factor (EGF) to enhance the development of murine preimplantation embryos cultured in vitro. Two-cell stage mouse embryos were cultured for 72 hr with EGF at concentrations of 0.1 to 100 ng/ml. The percentage of embryos which developed to the expanded and hatching blastocyst stage at 72 hr was determined. RESULTS: The percentage of fully expanded and hatching murine blastocysts at 72 hr was significantly higher following incubation with EGF at concentrations of 2 ng/ml (44 +/- 4.1%; P < 0.02), 20 ng/ml (41 +/- 3.2%; P = 0.04), 50 ng/ml (43 +/- 2.5%; P < 0.04), and 100 ng/ml (46 +/- 3.6%; P = 0.001) compared to controls. This effect of enhanced embryonic development by EGF was neutralized by coincubation with 1.0 micrograms/ml of anti-EGF antibody. CONCLUSION: EGF at concentrations of 2 to 100 ng/ml significantly enhanced the percentage of expanded and hatching murine blastocysts at 72 hr.

Animals↗

Functional roles of insulin and insulinlike growth factors in preimplantation mouse embryo development.

Growth factors are known to play important roles in cellular proliferation and differentiation. However, little information is available concerning their roles in the earliest stages of mammalian development. The effect of physiologic levels of insulin, insulinlike growth factor-I, and insulinlike growth factor II (IGF-I and -II) on DNA, RNA, and protein synthesis in preimplantation stages of the mouse are described in this study. Quantitative studies of the incorporation of labeled thymidine, uridine, and methionine into trichloroacetic acid-insoluble material by different developmental stages of preimplantation mouse embryos labeled in vitro, indicate that physiologic levels of insulin stimulated DNA, RNA, and protein synthesis with significant effects observed first at the morula stage of development. In contrast, neither IGF-I nor IGF-II stimulated DNA, RNA, or protein synthesis to a significant degree under the same experimental conditions. These results suggest a functional role for insulin at the earliest stages of mammalian embryogenesis.

Animals↗

A simplified technique for embryo biopsy: use of the same micropipette for zona drilling and blastomere aspiration.

PURPOSE: Using different micropipettes for zona drilling and blastomere aspiration for embryo biopsy is prevalent at centers of preimplantation genetic diagnosis. The purpose of our study was to simplify the technique by using only one micropipette. METHODS: In this animal model, ICR mouse embryos at the four-cell stage (n = 446) were randomly allocated into two groups; a biopsied group (n = 224) for blastomere aspiration and a control group (n = 222) without micromanipulation. We used a drilling/biopsy micropipette to drill a hole in the zona by expulsion of acidified Tyrode's solution and to aspirate the blastomere by gentle suction with the same micropipette and pull it out of the zona. One blastomere was biopsied from each embryo. RESULTS: In all, 222 (99.1%) intact blastomeres were successfully biopsied from 224 embryos. Only two blastomeres were damaged during aspiration. The capacity for blastocyst development (92.4 vs 93.7%) was not different between the two groups, but the percentages of embryos hatching (51.8 vs 18.0%) and hatched (29.9 vs 8.1%) were significantly higher in the biopsied group than in the control group. CONCLUSIONS: This simplified technique of embryo biopsy is safe and highly efficient for obtaining blastomeres for preimplantation genetic diagnosis and may also facilitate hatching of the blastocysts.

Animals↗

Molecular regulation of neural crest development.

The neural crest is a transient embryonic structure that gives rise to a multitude of different cell types in the vertebrate. As such, it is an ideal model to study the processes of vertebrate differentiation and development. This review focuses on two major questions related to neural crest development. The first question concerns the degree and time of commitment of the neural crest cells to different cell lineages and the emerging role of the homeobox containing genes in regulating this process. Evidence from the cephalic crest suggests that the commitment process does start before the neural crest cells migrate away from the neural tube and gene ablation experiments suggest that different homeobox genes are required for the development of neural and mesenchymal tissue derivatives. However, clonal analysis of neural crest cells before migration suggests that many of the cells remain multi-potential indicating that the final determinative steps occur progressively during migration and in association with environmental influences. The second question concerns the nature of the environmental factors that determine the differentiation of neural crest cells into discrete lineages. Evidence is provided, mainly from in vitro experiments, that purified growth factors selectively promote the differentiation of neural crest cells down either sympathetic, adrenal, sensory, or melanocytic cell lineages.

Animals↗

HSP gene expression and HSF2 in mouse development.

During the pre-implantation phase of development, the mouse embryo synthesizes HSC70, and HSP90 alpha and beta at a very high rate. After implantation, the expression of HSPs appears non-coordinated and is not uniform in the different tissues. The expression of inducible HSPs appears later in development than that of constitutive members of the family. HSP25 is highly expressed early in heart and muscle development, but also in some structure of the central nervous system. HSC70 and HSP90 beta are expressed ubiquitously, but their expression reaches very high levels in the nervous system (neural tracks) and during bone morphogenesis (in the hypertrophic chondrocytes). The mechanisms involved in HSP expression during mouse embryogenesis are probably diverse, involving tissue-specific sequences. Although the DNA-binding activity and expression of the second heat shock transcription factor, HSF2, seems to be developmentally regulated, becoming detectable at the blastocyst stage and reaching a peak at day 10 of development, there is no obvious correlation between the level of this factor and the expression of HSPs. HSF2 might be involved in the onset of expression of HSPs, regulate (inhibit) their expression, or control the expression of other developmental genes yet to be discovered.

Animals↗

Carboxypeptidases from A to z: implications in embryonic development and Wnt binding.

Carboxypeptidases perform many diverse functions in the body. The well-studied pancreatic enzymes (carboxypeptidases A1, A2 and B) are involved in the digestion of food, whereas a related enzyme (mast-cell carboxypeptidase A) functions in the degradation of other proteins. Several members of the metallocarboxypeptidase gene family (carboxypeptidases D, E, M and N) are more selective enzymes and are thought to play a role in the processing of intercellular peptide messengers. Three other members of the metallocarboxypeptidase gene family do not appear to encode active enzymes; these members have been designated CPX-1, CPX-2 and AEBP1/ACLP. In this review, we focus on the recently discovered carboxypeptidase Z (CPZ). This enzyme removes C-terminal Arg residues from synthetic substrates, as do many of the other members of the gene family. However, CPZ differs from the other enzymes in that CPZ is enriched in the extracellular matrix and is broadly distributed during early embryogenesis. In addition to containing a metallocarboxypeptidase domain, CPZ also contains a Cys-rich domain that has homology to Wnt-binding proteins; Wnts are important signaling molecules during development. Although the exact function of CPZ is not yet known, it is likely that this protein plays a role in development by one of several possible mechanisms.

Amino Acid Sequence↗