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PubMed2004

Immunohistochemical localization of peroxisomal enzymes during rat embryonic development.

Peroxisomes are cytoplasmic organelles involved in a variety of metabolic pathways. Thus far, the morphological and biochemical features of peroxisomes have been extensively characterized in adult tissues. However, the existence of congenital peroxisomal disorders, primarily affecting tissue differentiation, emphasizes the importance of these organelles in the early stages of organogenesis. We investigated the occurrence and tissue distribution of three peroxisomal enzymes in rat embryos at various developmental stages. By means of a highly sensitive biotinyl-tyramide protocol, catalase, acyl-CoA oxidase, and ketoacyl-CoA thiolase were detected in embryonic tissues where peroxisomes had not thus far been recognized, i.e., adrenal and pancreatic parenchyma, choroid plexus, neuroblasts of cranial and spinal ganglia and myenteric plexus, and chondroblasts of certain skeletal structures. In other tissues, i.e., gut epithelium and neuroblasts of some CNS areas, they were identified earlier than previously. In select CNS areas, ultrastructural catalase cytochemistry allowed identification of actively proliferating organelles at early developmental stages in several cell types. Our data show that in most organs maturation of peroxisomes parallels the acquirement of specific functions, mainly related to lipid metabolism, thus supporting an involvement of the organelles in tissue differentiation.

Acetyl-CoA C-Acyltransferase↗
PubMed1989

Effects of L-asparaginase on rat embryonic development and yolk sac membrane in vitro.

A comparative analysis of the teratogenic effects of L-asparaginase on 10.5- and 11.5-day rat embryos after 24 and 48 hours of exposure in vitro, respectively, were performed. Several medium concentrations of L-asparaginase (0.05, 0.25, and 1.5 IU/ml) were tested in both embryo series. Resulting embryos were submitted to morphological studies in a search for a specific route of pathogenesis. Morphological alterations of the visceral yolk sac were also studied to investigate its contribution to L-asparaginase teratogenicity in rats. Main embryonic malformations (open truncal neural tube, open encephalic vesicles, anophthalmia, lack of inversion, abnormal frontolateral protrusions, great vascular dilations at the cephalic level) and developmental retardation were already generated after the first 24 hours of culture (embryos of 10.5 days) and presented a dose-response relationship. Vascular dilations and neurulation disturbances seemed to be related to an early mesenchyme deficiency. Reduced number of mesenchymal cells was more evident in embryos of 10.5 days than those of 11.5 days, suggesting the existence of a later compensatory mechanism of cellular proliferation in the older embryo. Visceral yolk-sac endodermal cells at both embryonic stages were greatly deformed and enlarged by an increase of the high electron-dense vacuolar system. Therefore, both a blockage of the processes of lysosomal digestion and derived trophic deficiencies probably existed. A double teratogenic mechanism for L-asparaginase is postulated: a direct action mainly in younger embryos (before invagination of the embryo into the yolk sac) and a yolk sac-mediated one.

Analysis of Variance↗
PubMed1993

Early embryonic development in prolific Meishan pigs.

Prenatal mortality in European pigs is estimated at 30-40%, the majority of which occurs between days 12 and 18 after mating. Chinese Meishan pigs are prolific, averaging three to five more pigs per litter than do European breeds. Early reports into the fecundity of Meishan females suggested that their prolificacy resulted from lower embryonic mortality when compared with European females exhibiting the same ovulation rate. The preponderance of evidence suggests that there are no differences between Meishan and European breeds in either morphological embryo diversity within a litter or embryo mortality before day 12 after mating. Recent studies from our laboratory suggest that preimplantation embryos from Meishan females exhibit markedly reduced growth rates and oestrogen secretory activities through day 12 when compared with embryos from Yorkshire females. The significantly reduced conceptus sizes of Meishan versus European breeds on day 30 of gestation provide additional evidence of the reduced growth rate of Meishan embryos. Furthermore, because embryonic oestrogen production is known to alter uterine secretion of histotroph, the lower oestrogen production by Meishan embryos in the Meishan uterus may result in more gradual alterations in the uterine environment that are beneficial for conceptus survival and subsequent litter size. Recent studies using cross-transfer of Meishan and Yorkshire embryos on day 2 after mating have led to the suggestion that there is a major effect of recipient genotype on embryonic growth rate and oestrogen secretion. In these studies, both Meishan and Yorkshire embryos transferred to Meishan uteri exhibited marked decreases in morphological development and oestrogen content on day 12 when compared with embryos transferred to Yorkshire recipients. These data suggest the presence of factor(s) in endometrial secretions from Meishan females that reduce the growth rate and oestrogen secretory potential of preimplantation conceptuses.

Animals↗
PubMed1990

A novel oncofetal gene is expressed in a stage-specific manner in murine embryonic development.

A novel cDNA clone obtained from a murine T-lymphoma library hybridizes to transcripts expressed in placenta and embryos (Pem) in a stage-specific manner. The Pem cDNA sequence predicts an intracellular hydrophilic protein with no significant sequence similarity to other DNA or protein sequences. Pem transcripts are abundant in 7- and 8-day mouse embryos, but decrease precipitously thereafter. On Day 9 they become abundant in placenta and yolk sac, persisting there until parturition. Although Pem transcripts are present in immortalized and tumorigenic cell lines from several different cell lineages, they are not detectable in any of 15 adult tissues tested. The expression of Pem during fetal development and its presence in immortalized and neoplastic cell lines is consistent with the properties expected of an "oncofetal" gene.

Amino Acid Sequence↗
PubMed2003

Pregnancies and improved early embryonic development with bovine oocytes matured in vitro with 9-cis-retinoic acid.

Retinoids have an important role in cell growth, morphogenesis and differentiation. In the present study the developmental potential of bovine oocytes was examined after in vitro maturation in the presence of 9-cis-retinoic acid, a vitamin A metabolite, at 5 nmol l(-1) in chemically defined conditions. Experiments studied early in vitro development, blastocyst differential cell counts and the capacity of embryos to establish pregnancy after transfer to recipients. After in vitro fertilization and culture in simple medium, blastocyst development and hatching rates increased in oocytes matured with 9-cis-retinoic acid. Addition of ethanol (used as a solvent for 9-cis-retinoic acid) resulted in higher cell counts and proportions of cells in the inner mass of day 7 blastocysts. Day 8 blastocysts represented most differences observed in the number of cells. In these embryos, ethanol and 9-cis-retinoic acid increased both the number of cells and proportions in the inner mass. However, while ethanol treatment reduced the number of cells in the trophectoderm, 9-cis-retinoic acid did not. The total number of cells was unaffected by treatment within 1 day, although untreated oocytes lead to day 8 blastocysts with reduced total cell counts. Once transferred to recipients, both fresh and vitrified-warmed blastocysts derived from oocytes matured with 9-cis-retinoic acid yielded more pregnancies at day 60. Modifications of retinoid metabolism affect development and trophectoderm differentiation, and in vitro maturation with 9-cis-retinoic acid increased the developmental competence of the oocyte.

Alitretinoin↗
PubMed1993

Analysis of embryonic development with a model for under- or overdispersion relative to binomial variation.

Observations from a study of the development of ovulations into embryos for Texel sheep are analysed with a model for count data that are under- or overdispersed relative to binomial variation. The analysis is based on maximum quasi-likelihood (McCullagh and Nelder, 1989, Generalized Linear Models, 2nd edition, London: Chapman and Hall), following an approach suggested by Williams (1982, Applied Statistics 31, 144-148). The dispersion parameter is developed as a combination of a variance component representing shared maternal effects and a correlation, typically negative, between ovulations within ewes. The number of ovulations (the binomial denominator) is included as an explanatory variable.

Androstenedione↗
PubMed1998

The hypoxia-responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development.

Mice lacking the hypoxia-inducible transcription factor EPAS1 die at mid-gestation. Despite normal morphological development of the circulatory system, EPAS1-deficient mice display pronounced bradycardia. In addition to the vascular endothelium, EPAS1 is expressed intensively in the organ of Zuckerkandl (OZ), the principle source of catecholamine production in mammalian embryos. EPAS1-deficient embryos contained substantially reduced catecholamine levels. Mid-gestational lethality was rescued by administration of the catecholamine precursor DOPS to pregnant females. We hypothesize that EPAS1 expressed in the OZ senses hypoxia during mid-gestational development and translates this signal into an altered pattern of gene expression, leading to increases in circulating catecholamine levels and proper cardiac function.

Animals↗
PubMed1990

Novel approach to evaluate the interaction of pulsed ultrasound with embryonic development.

An in vitro whole-embryo culture system was used which allowed ultrasound to interact directly with rat embryos, at 9.5 days of gestation, under conditions of controlled temperature. Neural plate damage, expressed during a critical period of forebrain development, was evaluated 48 h post-insonation. Ultrasound-induced effects were thus identified in the absence of significant temperature changes or complications from the influence of maternal physiology. Exposures to 3.2 microseconds pulses of 3.14 MHz ultrasound at a PRF of 2 kHz and 1.2 W cm-2 intensity (ISPTA) for durations of 5, 15 or 30 min produced no major morphological abnormalities at a temperature of 38.5 degrees C. Embryonic response to stress was evidenced by changes in protein synthesis, and delayed development was indicated by a reduction in somite number. These effects were enhanced when the insonation temperature was elevated by 1.5 degrees C.

Animals↗
PubMed1981

Spatiotemporal patterns of fibronectin distribution during embryonic development. II. Chick branchial arches.

It has recently been demonstrated with the chick limb in ovo that successive nonrandom patterns of fluorescent staining with specific antibodies to fibronectin indicate the cartilagenous primordia prior to overt chondrogenesis. Given the apparent nonequivalence of embryonic cartilages, the purpose of this study was to determine whether this phenomenon was unique to developing chick limbs or is a more general characteristic of chondrogenesis. The appearance and distribution of fibronectin during chick first and second branchial arch development in ovo was investigated by indirect immunofluorescence techniques in H.H. stages 15-26. Fibronectin can be detected in early stages in areas presumed to be composed mainly of ectomesenchyme. During later stages of development, successive nonrandom patterns of fibronectin distribution appear to precede overt chondrogenesis as demonstrated by alcian blue staining. Pretreatment of cartilage with testicular hyaluronidase, prior to fibronectin staining, revealed that fibronectin was still present, suggesting that it was masked by proteoglycans. Fibronectin was also detected in the developing membrane bones of the mandible. The nonrandom patterning of fibronectin distribution in ovo in chick branchial arches and limb buds, respectively derived from neural crest and somatic mesoderm, were similar. It appears that specific patterns of fibronectin distribution were characteristic of chondrogenesis, regardless of the embryonic origin of the cartilage. This phenomenon may prove to be an extremely useful probe for early developmental skeletal abnormalities.

Animals↗
PubMed1999

Targeted ablation of gonadotrophs in transgenic mice affects embryonic development of lactotrophs.

Ablation of pituitary gonadotrophs was obtained in transgenic mice expressing diphtheria toxin A (DTA) under control of the -313/+48 bovine glycoprotein hormone alpha-subunit (alphaSU) promoter, previously shown to be active in mouse gonadotrophs but not in thyrotrophs. Development of hormone-producing cell types was assessed on the day of birth by computer-assisted image analysis on paraffin-embedded, immunostained pituitary sections. Six out of 50 transgenic F0 ('founder') mice (3 males and 3 females) showed a nearly complete disappearance of gonadotrophs but not of thyrotrophs. The number of lactotrophs and the relative area occupied by PRL-immunoreactivity were significantly reduced in the gonadotroph-depleted mice. The size of lactotroph clusters was smaller in the absence of gonadotrophs. The number and immunoreactive area of corticotrophs and somatotrophs, on the other hand, were not significantly affected by gonadotroph ablation. Based on the reported evidence that fetal ovaries do not produce steroid hormones as a result of lack of expression of at least three of the steroidogenic enzymes, P450scc, P450c17, and P450arom, the present observations can hardly be explained by a decline in estrogen levels due to gonadotroph ablation. Rather, the present data indicate that gonadotrophs directly stimulate the development of lactotrophs during fetal and early postnatal life, consistent with previous in vitro observations, and/or that gonadotrophs may share a cell-lineage relationship with a subpopulation of lactotrophs.

Animals↗
PubMed1997

Expression of the acatalytic carbonic anhydrase VIII gene, Car8, during mouse embryonic development.

The carbonic anhydrase (CA)-like protein, CA VIII, lacks the typical carbon dioxide hydrase activity of the CA isozymes. However, the high degree of amino acid sequence similarity between the products of the mouse and the human CA VIII genes suggests an important biological function. We have attempted to investigate the function of this gene in mammalian development by conducting an in situ hybridization study on sagittal sections of mouse embryos at gestation days of 9.5-16.5 using a 35S-labelled riboprobe. Results indicate that this gene (called Car8 in mice) is expressed as early as day 9.5 in a variety of organs including liver, branchial arches, neuroepithelium and developing myocardium. Between days 10.5 and 12.5, it showed a widespread distribution of mRNA expression that became more restricted as development progressed. The level of expression of Car8 mRNA was relatively high in the brain, liver, lung, heart, gut, thymus and epithelium covering the head and the oronasal cavity.

Animals↗
PubMed2000

Lysyl oxidase and P-ATPase-7A expression during embryonic development in the rat.

Lysyl oxidase activity is critical for the assembly and cross-linking of extracellular matrix proteins, such as collagen and elastin. Moreover, lysyl oxidase activity is sensitive to changes in copper status and genetic perturbations in copper transport, e.g., mutations in the P-type ATPase gene, ATP7A, associated with cellular copper transport. Lysyl oxidase may also serve as a vehicle for copper transport from extracellular matrix cells. Herein, we demonstrate that sufficient lysyl oxidase functional activity is present in the rat embryo at gestation day (GD) 9 to be detected in conventional enzyme assays. Estimation of embryonic lysyl oxidase functional activity, however, required partial purification in order to remove inhibitors. From GD 9 to GD 15, lysyl oxidase activity was relatively constant when expressed per unit of protein or DNA. In contrast, the steady-state levels of lysyl oxidase and ATP7A mRNA, measured by RT-PCR and expressed relative to total RNA and cyclophilin mRNA, increased approximately fourfold from GD 9 to 15. The pattern of temporal expression for ATP7A was consistent with its possible role in copper delivery to lysyl oxidase.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Changes in the prekeratin set and in the intracellular distribution of intermediate filaments during the embryonic development of the liver in the rat].

Monoclonals against three individual proteins of the epithelial intermediate filaments, prekeratins (PK40, PK49, PK55), were used for immunofluorescence studies of the cryostat sections of the rat embryonic liver. The dynamics of expression and intracellular distribution of prekeratins reflects that of morphological rearrangement of the liver. The development of the system of liver beams was accompanied by changes in the expression and intracellular distribution of PK49 and PK55 and the development of the system of bile ducts by changes in all three PKs. From day 20-21 of embryogenesis all three PKs are expressed in cholangiocytes, while PK49 and PK55 in hepatocytes only.

Animals↗
PubMed1998

In vitro assessment of the effect of halogenated hydrocarbons: chloroform, dichloromethane, and dibromoethane on embryonic development of the rat.

Halogenated hydrocarbons are widely used in industry, the laboratory, and in the home. In the present study three of these solvents--chloroform, dichloromethane, and dibromoethane--were examined for embryotoxic/teratogenic potential using rat embryo culture. The results showed that each of the solvents had a concentration-dependent embryotoxic effect on the developing rat embryo in vitro. The effect and no-effect concentrations (expressed in mumol/ml culture medium), respectively, for each of the halogenated hydrocarbons tested were: dibromoethane--0.33, < 0.18; chloroform--2.06, 1.05; dichloromethane--6.54, 3.46. The levels of chloroform and dichloromethane found to be embryotoxic in the present study were compared to reported blood levels attained following controlled human exposure. In the industrial situation, if the current exposure levels are adhered to, chloroform and dichloromethane appear to have little potential for reproductive toxicity in the human. Fatal or near fatal solvent levels would be required in the mother for the embryotoxic level to be reached. For dibromoethane, there are no reports following controlled human exposure presumably due to its carcinogenicity. In an attempt to elucidate the mechanism of embryotoxicity, histological studies were performed after exposure of rat embryos to an embryotoxic level of each of the halogenated hydrocarbons studied, for increasing time periods up to the standard 40-hour culture. Marked cell death in the neuroepithelium of the developing neural tube was a prominent feature in all embryos exposed to an embryotoxic level of these solvents for periods of 16 hours of longer.

Animals↗
PubMed2004

Isolation of a library of target-sites for sequence specific DNA binding proteins from chick embryonic heart: a potential tool for identifying novel transcriptional regulators involved in embryonic development.

Enormity of the metazoan genomes and divergence in their regulation impose a serious constraint on the comprehensive understanding of context specific gene regulation. DNA elements located in the promoter, enhancer, and other regulatory regions of the genome dictate the temporal and spatial patterns of gene activities. However, owing to the diminutive and variable nature of the regulatory DNA elements, their identification and location remains a major challenge. We have developed an efficient strategy for isolating a repertoire of target sites for sequence specific DNA binding proteins from embryonic chick heart. A comprehensive library of such sequences was constructed and authenticated using various parameters including in silico determination of functional binding sites. This approach, therefore, for the first time, established an experimental and conceptual framework for defining the entire repertoire of functional DNA elements in any cellular context.

Amino Acid Sequence↗
PubMed2003

Actions of tumor necrosis factor-alpha on oocyte maturation and embryonic development in cattle.

PROBLEM: Infertility can accompany mastitis in cattle. Involvement of tumor necrosis factor-alpha (TNF-alpha) in this phenomenon is suggested by observations that circulating concentrations of TNF-alpha are elevated after intramammary infection or infusion of endotoxin. It was hypothesized that (1) TNF-alpha acts on the oocyte during maturation to decrease the percent of oocytes that cleave and develop following fertilization; (2) exposure of embryos to TNF-alpha after fertilization reduces development to the blastocyst stage; and (3) TNF-alpha increases the proportion of blastomeres that undergo apoptosis in a stage-of-development dependent manner. METHOD OF STUDY: In one experiment, oocytes were matured with various concentrations of TNF-alpha and then fertilized and cultured without TNF-alpha. In another study, embryos were cultured with TNF-alpha for 8 days beginning after fertilization. Finally, embryos were collected at the two or four-cell stage (at 28-30 hr after insemination) or when > or = 9-cells (at day 4 after insemination) and cultured +/- TNF-alpha for 24 hr. The proportion of blastomeres undergoing apoptosis was then determined by the TUNEL procedure. RESULTS: Addition of TNF-alpha to maturation medium did not affect the proportion of oocytes that cleaved. However, the percent of oocytes that developed to the blastocyst stage at day 8 after insemination was reduced (P = 0.05) at all TNF-alpha concentrations tested (0.1-100 ng/mL). When added during embryo culture, there was no significant effect of TNF-alpha on the proportion of oocytes that became blastocysts. In addition, TNF-alpha did not induce apoptosis in two and four-cell embryos. For embryos > or = 9-cells, however, 10 and 100 ng/mL TNF-alpha increased (P < 0.05) the percent of blastomeres labeling as TUNEL-positive. CONCLUSION: TNF-alpha can have deleterious actions on oocyte maturation that compromise development of the resultant embryo. While exposure of fertilized embryos to TNF-alpha did not inhibit development to the blastocyst stage, TNF-alpha increased the percentage of blastomeres undergoing apoptosis when exposure occurred for embryos > or = 9-cells. Increased blastomere apoptosis could conceivably compromise subsequent embryo survival.

Animals↗
PubMed1985

Influence of egg storage on hatchability, embryonic development and vitamin status in hatching broiler chicks.

Storage of eggs for 14 d prior to incubation depressed hatchability by 10%. Most of the increase in embryonic mortality (64%) occurred during the first week of incubation, with a further 30% taking place during the third week. Chicks hatching from stored eggs did not show any increased incidence of physical abnormalities characteristic of vitamin deficiencies. Chicks from stored eggs showed haematological abnormalities characteristic of mild macrocytic anaemia. Such changes can result from deficiencies of folic acid or cobalamin. Biochemical and haematological data did not suggest impairments in the status of the chicks with respect to a range of other vitamins.

Animals↗