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PubMed1995

Effects of folate deficiency on embryonic development.

While there is strong evidence that folate deficiency including the use of anti-folate drugs in early pregnancy is teratogenic and may lead to a range of serious abnormalities of the developing fetus including intrauterine death, the mechanism(s) for these effects have not yet been delineated. In neural tube defects, there is increasing evidence that marginal folate status exacerbates the effect of an underlying genetic defect in the mother, the fetus, or both. An abnormal relationship between the ingestion of folate and the folate levels in red blood cells has been found in women who have given birth to infants with neural tube defects. Periconceptional folate supplementation has been shown to give effective protection against the development of neural tube defects. The mechanism of the prevention is as yet unknown. However, folic acid will not prevent all cases of neural tube defect. Moreover, neither determinations of periconceptional vitamin profiles (Mooij et al, 1993) nor determinations of methylmalonic acid and homocysteine levels will detect all women at risk. Anencephaly and spina bifida can be identified prenatally by detection of excessive levels of alpha-fetoprotein in amniotic fluid and maternal serum and by ultrasonographic scanning (Wilson and Rudd, 1993). Both genetic counselling and prenatal diagnosis should be offered to women who are recognized to be at high risk. Research into the underlying biology of neural tube defects is of major importance. The identification of underlying genetic defects would allow for genetic testing and better counselling of families at risk for the occurrence of a neural tube defect.

Animals↗

PubMed1994

Embryonic development of the stomatogastric nervous system in Drosophila.

Using several cell-specific markers, the pattern of proliferation, morphogenesis, and neuronal differentiation of the Drosophila larval stomatogastric nervous system (SNS) was analyzed. In the late embryo, four SNS ganglia (frontal ganglion, hypocerebral ganglion, paraesophageal ganglion, ventricular ganglion) can be distinguished. In the early embryo, the precursor cells of the SNS (SNSPs), being an integral part of the anlage of the esophagus, undergo four synchronous rounds of division. Subsequently, SNSPs segregate from the esophageal epithelium in a complex and stereotyped pattern. The majority of SNSPs invaginate and transiently form three (rostral, intermediate, caudal) pouches that, after separating from the esophagus, become epithelial vesicles. At later stages, these SNSPs gradually lose their epithelial phenotype. Starting at the anterior-dorsal tip of each vesicle, SNSPs dissociate from one another and migrate to the various locations where they differentiate as neurons. Cells of the rostral and intermediate vesicle contribute to the frontal ganglion; the hypocerebral ganglion develops from the intermediate vesicle, the paraesophageal ganglion from the rostral vesicle, and the ventricular ganglion from the caudal vesicle. In addition to the invaginating SNSPs, several distinct groups of SNSPs delaminate as individual cells from the esophageal epithelium. Three clusters of SNSPs delaminate from a region anterior to the rostral pouch; a single SNSP delaminates from the tip of each pouch. All delaminating SNSPs contribute to the frontal ganglion. A significant number of SNSPs undergo cell death. In the late embryo, the stomatogastric ganglia are interconnected by the recurrent nerve and esophageal nerves. The frontal ganglion projects to the brain via the frontal connectives. Both recurrent nerve and frontal connectives are pioneered by small subpopulations of early differentiating stomatogastric neurons that most likely derive from among the dSNSPs and iSNSPs.

Animals↗

PubMedDate not supplied

Mouse genetics provides insight into folliculogenesis, fertilization and early embryonic development.

After colonization of the gonad, mouse female germ cells enter into the prophase of the first meiotic division as a mid-gestational hallmark of gender. Perinatally, oocytes interact with granulosa cells to form primordial follicles which, with cyclic periodicity, enter into a 3-week growth phase that culminates in meiotic maturation and ovulation. Successful fertilization in the oviduct results in the onset of embryogenesis. Genes expressed in oocytes encode maternal factors that control many of these developmental processes. The establishment of mouse models in which specific genes have been disrupted offers robust insights into molecular mechanisms that control oogenesis, folliculogenesis, fertilization and early embryogenesis. Although relatively few developmental circuits have been characterized in genetic detail, the ongoing revolution in mouse genetics holds great promise. These model systems provide novel information into the molecular basis of the pathways required for oocyte-specific processes as well as for interactions with the temporally changing environment of female germ cells. The similarities between the mouse and human genomes provide assurance that this knowledge will rapidly translate into a better understanding of human reproduction.

Animals↗

PubMed1988

Effect of diclofenac on implantation and embryonic development in the rat.

Recent evidence suggests that decidualization can be considered a modified inflammatory reaction. In this work we intended to determine whether the prostaglandin inhibitor diclofenac could affect implantation. Rat blastocysts were cultured in diclofenac in vitro, then implanted to host mothers on day 5 of pseudopregnancy. Large doses of diclofenac in culture were toxic. Smaller doses had a profound effect on implantation. Another group of host mothers received diclofenac i.p., one hour prior to transfer of untreated blastocysts. The results were compared to parallel controls without diclofenac treatment. Control animals had a 72% implantation rate, whereas there was only a 35-41% implantation rate after in vitro diclofenac treatment. In the treated host mothers only 7% of embryos were normal, while 34% were growth-retarded. More normal embryos were found when the blastocysts were cultured with diclofenac. The observations indicate that diclofenac administered to the mother inhibits the ongoing process of implantation and placentation, whereas following in vitro exposure the embryo can recover from the insult. The implications for a better understanding of the process of implantation are discussed.

Animals↗

PubMed1989

Embryonic development after microsurgical repair of polyspermic human zygotes.

Correction of polyspermy through pronucleus extraction in the absence of membrane relaxants was applied to 25 polyspermic human zygotes. Nine zygotes survived the procedure, and seven cleaved normally (two of which were fixed for chromosome analysis); two proceeded to compact and one cavitated. Eighteen of the polyspermic zygotes (14 with three pronuclei and 4 with four pronuclei) were obtained from zona pellucida-intact oocytes, and seven (1 five-pronucleate 3 four-pronucleate, and 3 three-pronucleate) from previously zona-drilled oocytes. Survival and cleavage occurred in all groups except in four- and five-pronucleate zona-drilled zygotes. Criteria used to identify male pronuclei were (1) pronucleus-associated sperm tails, (2) increased pronucleus size, and (3) greater distance (relative to female pronuclei) from the second polar body. Sperm tails were never seen and pronucleus size usually was identical. Therefore, the third criterion was used, although its reliability should be further evaluated. Until complete pronucleus removal techniques and reliable pronucleus selection criteria are perfected, embryo replacement after polyspermy correction could result in aneuploidy and molar pregnancy.

Embryonic and Fetal Development↗

PubMed1993

Nuclear control of early embryonic development in domestic pigs.

In mammals, growing oocytes have characteristically high levels of RNA synthesis. After the initiation of meiosis, that is germinal vesicle breakdown, this RNA synthesis ceases. Although there is limited evidence for RNA synthesis by the zygote, significant amounts of RNA synthesis do not occur until a species-specific cell stage. In pigs, significant amounts of mRNA synthesis cannot be detected before the four-cell stage. There appear to be three qualitatively different periods of transcription during the four-cell stage. The first occurs during a short (< 2 h) G1 phase. The second, occurs after completion of DNA synthesis (S phase) about 16 h after cleavage to the four-cell stage, and the third occurs about 24 h after cleavage to the four-cell stage. Correlated with these changes in RNA synthesis are changes in nucleolar morphology, amino acid transport characteristics, protein production, mitochondrial morphology, and metabolism of the embryo. The mechanisms that regulate initiation of RNA synthesis in early mammalian embryos appear to repress transcription. A state of transcription permissiveness then follows that sets into motion the differentiation programme.

Amino Acids↗

PubMed1994

Early embryonic development in the Djungarian hamster (Phodopus sungorus) is accompanied by alterations in the distribution and intensity of an estrogen (E2)-dependent oviduct glycoprotein in the blastomere membrane and zona pellucida and in its association with F-actin.

The luminal environment of the estrogen (E2)-dominated mammalian oviduct generates and sustains the environment in which the first embryonic cleavages take place. The objective of this study was to determine, by use of an antiserum against an E2-dependent sheep oviduct secretory glycoprotein (M(r) 90,000-92,000), whether the E2-dominated and pregnant oviduct of the Djungarian hamster (Phodopus sungorus) releases an antigenically related protein. If the protein was present, a secondary objective was to define its fate and association with filamentous-actin (f-actin) and chromatin patterns in early cleavage-stage embryos. Oviduct flushings containing embryos (1-cell fertilized, 2-, 4-, and 8-cells), and uterine flushings (> 16 cell embryos) were obtained from pregnant hamsters. Embryos were removed from flushings, and oviduct secretions were analyzed by Western blotting. The zona pellucida was removed with acid Tyrode's solution from approximately half of the 2-, 4-, and 8-cell embryos. Zona-intact and zona-free embryos were then fixed and subjected to triple immunofluorescence staining with an antiserum to the sheep oviduct protein, rhodamine phalloidin, and Hoechst 33258. An antigenically related protein M(r) 200,000) was detected in oviduct secretions of E2-treated, ovariectomized, and pregnant hamsters, and not in secretions from ovariectomized controls. In the zona pellucida of 1- and 2-cell embryos, the oviduct protein displayed an intertwining, reticular organization that was replaced by a diffuse and more intense accumulation in 4-, 8-, and > 16-cell embryos. In 2-cell embryos, punctate foci of the oviduct protein were distributed unevenly over the apical blastomere plasma membrane, forming patches in regions of f-actin exclusion, which were absent at later development stages. At the 4- and 8-cell stage of development, as blastomeres lost their spherical form by minimizing intercellular spaces, the oviduct protein took on a polarized arrangement and was intensely concentrated on membrane areas involved in cell-cell contact that were also the focus of f-actin. These data show that in early cleavage-stage hamster embryos, the intensity and pattern of staining for an E2-dependent oviduct protein (M(r) 200,000) that is released into the oviduct lumen during embryo transport can be distinguished on the basis of membrane f-actin display and blastomere number and shape. These events may mediate cellular processes related to blastomere cleavage, shaping, and/or adhesion that occur in the oviduct.

Actins↗

PubMed2001

Inactivation of the murine pyruvate dehydrogenase (Pdha1) gene and its effect on early embryonic development.

A deficiency of pyruvate dehydrogenase complex (PDC) in humans results in lactic acidosis and neurological dysfunction that frequently results in death during infancy. Using gene targeting technology, a silent mutation was introduced into the murine X-linked Pdha1 gene that encodes the alpha subunit of the pyruvate dehydrogenase or E1 component of the complex. Two loxP sequences were introduced into intronic sequences flanking exon 8 to generate the Pdha1(flox8) allele. In vitro studies in embryonic stem cells demonstrated that deletion of exon 8 ablated PDC activity. Homozygous Pdha1(flox8) females were bred with male mice carrying a wild-type Pdha1 allele and a transgene that ubiquitously expresses the Cre recombinase to produce progeny with a deletion in exon 8, Pdha1(Deltaex8). The majority of progeny were found to be mosaic with the presence of both the flox and deleted alleles, and there were no apparent phenotypic effects associated with the null allele. The mosaic mice were interbred to increase the degree of mosaicism for the Pdha1(Deltaex8) allele in the subsequent generation, resulting in a significantly smaller litter size (54% reduction). Embryos carrying predominantly the Pdha1(Deltaex8) allele were found to be globally delayed in development by 9.5 days postcoitus, with resorption occurring over the following several days. These findings demonstrate an essential role for oxidative metabolism of glucose during the early postimplantation period of prenatal development.

Alleles↗

PubMed1988

[Action of the paternal genome at the beginning of embryonic development].

Recent experiments using nuclear transfer between mouse embryos shows that the parental genomes are not equivalent: both of them must be present at least in a part of the blastomeres to allow full term development of the embryo. The paternal genome is preferentially involved in the development of trophoectodermic cells and appears to be eliminated from the primitive ectoderm when kept alone in androgenetic cells aggregated with normal ones. Differences of expression between parental genomes affect not only the X chromosome, but also several autosomes as evidenced by genetic analysis using meiotic non-disjunction. The nature of the imprinting mechanism that leads to the modification of parental homologous chromosomes is unknown. From experiments involving a particular strain of mice called DDK it appears that the egg cytoplasm may exert a specific action not only on the male pronucleus at the one-cell stage, but also on the paternal contribution of the diploid nucleus of the embryo at the 2-cell stage; a paternal developmental effect on the cytoplasm of one-cell stage embryo can also be demonstrated. These results show that nucleocytoplasmic interactions regulate early paternal gene expression differently from the maternal one.

Animals↗

PubMed1991

[Embryonal development of grouped lymphoid nodules (Peyer's patches) in the human ileum].

Embryogenesis of Payer's patches (PP) of the ileum, has been studied in 183 human fetuses 8-40-week-old. Their anlages appear on the 8th-9th week as an accumulation of atypical villi. At first the PP are localized in the cranial part of the ileum, and then spread caudally. Their most active increase in amount takes place from the 15th up to 17th weeks of development. From the 8th up to the 40th weeks the PP amount increase from 10 up to 37. During the same time their length develops from 0.7 up to 8.3 mm, their width--from 0.3 up to 2.2. The first lymphoid nodules++ in the PP are detected on the 14th week, then their number rises from 200 up to 3,500. The superficial area of all the PP, turning into the lumen of the ileum widens from 1.4 up to 620 mm2. Their predominate form is ellipsoid. During the whole prenatal period in the lymphoid nodules++ no germinative centers are revealed. Lymphocytes in the PP are identified in 8-9-week-old fetuses. By the 29th week the whole amount of lymphocytes in them increase up to 9.6 x 10(6) cells. Lymphocytic suspension of the PP of 8-9-week-old fetuses contains 1.7% of T-lymphocytes (E = POK) and 0.1% of B-lymphocytes (EAC = POK). By the 29th week their amount increases up to 9% and 7%, respectively, but by birth it does not reach their amount in the PP of mature organisms.

Embryonic and Fetal Development↗

PubMed1991

A model system and technique to study the effects of ultrasound irradiation on embryonic development.

To study the effects of ultrasound on development it is important to have a system which provides reliable results. We have designed a system which allows for reproducible irradiations of chick embryos in ovo. The irradiation system includes a heated sonation tank with ultrasound absorbers and a PC/AT computer-based data acquisition system for on-line monitoring of irradiations. The ultrasound detection microprobe and irradiation transducers were calibrated against an NBS traceable balance meter. An acoustic spacer was utilized to provide a more uniform profile of the irradiation beam. At the position of the embryo the ultrasound field geometry was determined. To maintain the chick embryo in its natural physiological state while minimizing ultrasonic reflections and standing-wave generation, two diametrically opposed windows were made in the eggshell along the ultrasound pathway and covered with polyethylene membranes. Using this irradiation system at intensity levels as high as 1.1 W/cm2 (spatial average, temporal average) for 10 min, the temperature rise is minimal.

Animals↗

PubMed1989

Combined effects of radiation and caffeine on embryonic development in mice.

The combined effect of radiation and caffeine has been studied in mouse embryos. Radiation and/or caffeine were administered to ICR mice on Day 11 of gestation. Intrauterine death, gross malformation, and fetal body weight were selected as indicators of effects. Doses of whole-body gamma irradiation were 0.5 to 2.5 Gy and those of caffeine were 100 and 250 mg/kg maternal body wt. Intrauterine mortality increased with increasing radiation dose; this trend was more remarkable in combination with caffeine. Gross malformations such as cleft palate and defects of forelegs and hindlegs appeared frequently in the fetuses treated with both radiation and caffeine. Decreased fetal weight was observed even in mice treated with 0.5 Gy of radiation or 100 mg/kg caffeine. There was a linear relationship between dose and reduction of fetal weight. The fetal weight was a sensitive, precise, and easy-to-handle indicator for the effects of growth retardation. Intrauterine mortality and frequencies of cleft palate and defects of forelegs and hindlegs were higher than the sum of those induced by radiation and by caffeine separately. The results indicated that the combined action of radiation and caffeine on intrauterine death and malformations was synergistic.

Abnormalities, Drug-Induced↗

PubMed2001

TGFbeta2 in corneal morphogenesis during mouse embryonic development.

To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.

Acetyltransferases↗

PubMed1987

Two human homeobox genes, c1 and c8: structure analysis and expression in embryonic development.

Two human cDNA clones (HHO.c1.95 and HHO.c8.5111) containing a homeobox region have been characterized, and the respective genomic regions have been partially analyzed. Expression of the corresponding genes, termed c1 and c8, was evaluated in different organs and body parts during human embryonic/fetal development. HHO.c1.95 apparently encodes a 217-amino acid protein containing a class I homeodomain that shares 60 out of 61 amino acid residues with the Antennapedia homeodomain of Drosophila melanogaster. HHO.c8.5111 encodes a 153-amino acid protein containing a homeodomain identical to that of the frog AC1 gene. Clones HHO.c1 and HHO.c8 detect by blot-hydridization one and two specific polyadenylylated transcripts, respectively. These are differentially expressed in spinal cord, backbone rudiments, limb buds (or limbs), heart, and skin of human embryos and early fetuses in the 5- to 9-week postfertilization period, thus suggesting that the c1 and c8 genes play a key role in a variety of developmental processes. Together, the results of the embryonic/fetal expression of c1 and c8 and those of two previously analyzed genes (c10 and c13) indicate a coherent pattern of expression of these genes in early human ontogeny.

Amino Acid Sequence↗

PubMed1996

Effect of cooling mouse spermatozoa to 4 degrees C on fertilization and embryonic development.

Attempts to freeze mouse spermatozoa in liquid nitrogen (-196 degrees C) have met with limited success. In an attempt to identify the factor(s) that damage mouse spermatozoa during cryopreservation, the effect of slow cooling to 4 degrees C was examined. Epididymal spermatozoa were collected into a variety of media at 37 degrees C, cooled slowly to 4 degrees C over 4 h and warmed in a water bath at 37 degrees C for 5 min. Survival of spermatozoa was assessed by motility, membrane integrity and acrosome status. Labelling with chlortetracycline showed that > 80% of spermatozoa were capacitated and had intact acrosomes immediately after warming compared with < 20% of freshly collected (control) spermatozoa. The rate of fertilization in vitro was similar using spermatozoa cooled in Dulbecco's phosphate-buffered saline and then mixed with oocytes immediately after warming and with control spermatozoa incubated for 2 h before mixing with oocytes (85%). Fewer oocytes were fertilized with spermatozoa cooled in either a modified HEPES-buffered Tyrode's medium or a simple HEPES-buffered medium with a high osmolarity (D3), 63% and 58%, respectively. Two-cell embryos were transferred to the oviducts of pseudopregnant recipients. Implantation was similar in all groups (81-88%) and 54-74% of embryos formed normal late stage fetuses.

Acrosome↗

PubMed1992

DM-20 mRNA is expressed during the embryonic development of the nervous system of the mouse.

We used both the polymerase chain reaction (PCR) and in situ hybridization to search for the presence of proteolipid protein (PLP) gene transcripts in the developing mouse. Total brain RNA extracted from 13-19-day embryos, analyzed by PCR, demonstrated the presence of a single transcript that was unambiguously identified with the DM-20 mRNA. RNA samples from postnatal day 2 animals also showed a signal corresponding to the PLP transcript, in addition to the DM-20 message. By in situ hybridization of 10-day embryos using a DM-20 antisense cRNA probe, we showed that the localization of the DM-20 message was restricted to the diencephalic basal plate. On the same embryo sections, in addition to the brain localization, an intense hybridizing signal was also detected in the trigeminal and spinal ganglia, the vagal glossopharyngeal ganglion, and the sympathetic ganglion chain. The demonstration of transcription of the PLP gene, long before the beginning of the myelination process, suggests that in addition to a structural function in myelin compaction, some of the products of the PLP gene (DM-20) may have a role during the compartmentalization and differentiation of the neural tube.

Animals↗

PubMed1995

DNA methylation and polyamines in embryonic development and cancer.

Mammalian DNA contains relatively large amounts of a modified base, 5-methyl-cytosine (m5C). Methylation of cytosine is catalyzed by DNA(cytosine-5)methyltransferase (DNA MTase). DNA methylation seems to play an important role in the regulation of gene expression during development. Thus, m5C may inhibit transcription by preventing the binding of transcription factors and/or by altering chromatin structure. The DNA methylation patterns of the male and female pronuclei are erased in the morula and early blastula, and when the blastocyst forms, most of the DNA has become demethylated. Following implantation, however, there is a surge of de novo methylation affecting the entire genome, and already by gastrulation DNA is methylated to an extent characteristic of that of the adult animal. During subsequent development, tissue-specific genes undergo programmed demethylation, which may cause their activation. Site-directed mutagenesis of the DNA MTase gene, has recently shown that DNA methylation is absolutely required for normal development of the early mouse embryo. DNA methylation and polyamine synthesis depend on a common substrate, S-adenosylmethionine (AdoMet). As a consequence, changes in cellular polyamine levels may affect the degree of DNA methylation. When the first step in the polyamine biosynthetic pathway is blocked, F9 teratocarcinoma stem cells accumulate large amounts of decarboxylated AdoMet, the aminopropyl group donor in polyamine synthesis, and go through terminal differentiation into parietal endoderm cells. The accumulation of decarboxylated AdoMet is a direct consequence of the polyamine-depleted state of the cell. Although the decarboxylated AdoMet molecule contains a methyl group, it does not act as a methyl group donor in DNA methylation. Instead it acts as a competitive inhibitor of DNA MTase. A consequence of polyamine depletion is therefore genome-wide loss of DNA methylation due to insufficient maintenance methylation during successive rounds of DNA replication. Our recent finding that prevention of the accumulation of decarboxylated AdoMet counteracts the differentiative effect lends further support to the hypothesis proposed.

5-Methylcytosine↗

PubMed1996

Fertilization, embryonic development and implantation of mouse oocytes with one or two laser-drilled holes in the zona, and inseminated at different sperm concentrations.

The zonae pellucidae of mouse oocytes were photo-ablated by an ultraviolet laser (337.1 nm) to create one or two 10 microm holes. The pulse energy used was approximately 3 microJ/s, with a frequency of 10 pulse/s. These laser zona-drilled (LZD) oocytes with one hole (LZD1) or two holes (LZD2) and the zona-intact controls were inseminated with spermatozoa at standard concentrations of 5x10(4), 5x10(5), 1x10(6) and 2x10(6)/ml. Fertilization was significantly improved at all sperm concentrations in LZD1 and LZD2 oocytes as compared to the controls. However, there was no significant difference in fertilization rates between LZD1 and LZD2. LZD2 produced significantly higher numbers of blastocysts at day 5. Hatching was significantly enhanced in the presence of either one or two holes in the zona. Polyploidy was generally absent, except in LZD2 oocytes (1%) inseminated at higher sperm concentrations. Differential cell counts of expanded LZD blastocysts were similar to those of the controls. Significantly fewer LZD2 blastocysts implanted and produced viable fetuses than LZD1 and control blastocysts. Morphological abnormalities of the fetuses were absent in all three groups. Laser zona-drilling using the ultraviolet laser was shown to be fast, efficient and safe.

Animals↗