An extensive numerical simulation of the cephalic furrow formation in Drosophila embryo

Here, we propose a new finite element model of the Drosophila embryo to simulate the cephalic furrow (CF) formation. Two different 3D geometries are introduced to reproduce the embryo. The two of them are parametrised through a novel system of curvilinear coordinates, well adapted for biological structures, which is obtained by three fictive electrostatic potentials. A gradient decomposition method is used to take into account both the active and the passive deformations occurring to the cells. Four simulations of the CF are proposed, which in turn considers singular and coupled aspects of the problem.     

Studies on the mechanism of different collagen glucosyltransferase reactions (Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum) in chick embryo liver.

1. The galactosylhydroxylysylglucosyltransferase (GGT) specific to collagen is located in the RER (rough endoplasmic reticulum), SER (smooth endoplasmic reticulum) and Golgi apparatus for the chick embryo liver. 2. The UDP-glucose collagen glucosyltransferase activities in chick embryo liver were solubilized by Nonidet P-40. 3. The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparation of Golgi apparatus CF2, smooth endoplasmic reticulum CF4 and rough endoplasmic reticulum CF8. 4. For the three fractions, data obtained in experiments were consistent with a sequential ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, collagen and UDP-glucose substrate, with different values for Km and Vmax.     

Changes in the Pattern of Intercellular Junctions during Early Embryogenesis of the Starfish, Asterias amurensis

Changes in the cellular adhesion pattern during the early embryogenesis of a starfish Asterias amurensis were examined using carboxyfluorescein (CF) dye as a probe. CF that was injected into one of the blastomeres at the 2- or 4-cell stage was in all cases restricted to the progeny cells of the CF-labelled blastomere. With the advancement of gastrulation, however, the injected dye was distributed not only to the progeny of the labelled blastomere, but also to cells that originated from non-injected blastomeres. At the beginning of mesenchyme cell release, the injected dye spread uniformly to most cells comprising the embryo. When one of the blastomeres situated in the vegetal hemisphere of an 8-cell embryo was labelled, the resulting embryo showed more intense fluorescence in the cells surrounding the archenteron than in the ectodermal layer, suggesting that the cells in ectodermal layer became associated more intimately or earlier than those surrounding the archenteron. Likewise, in double embryos formed by combining two denuded eggs, in which one egg had been labelled with CF, dye spread was observed when the ectodermal layer began to expand. The intercellular spread of CF dye in starfish embryo suggests that there is a dramatic change in the cellular adhesion pattern during the course of gastrulation.     

Long-term and transgenerational effects of cryopreservation on rabbit embryos

The short-term effects of cryopreservation and embryo transfer are well documented (reduced embryo viability, changes in pattern expression), but little is known about their long-term effects. We examined the possibility that embryo vitrification and transfer in rabbit could have an impact on the long-term reproductive physiology of the offspring and whether these phenotypes could be transferred to the progeny. Vitrified rabbit embryos were warmed and transferred to recipient females (F0). The offspring of the F0 generation were the F1 generation (cryopreserved animals). Females from F1 generation offspring were bred to F1 males to generate an F2 generation. In addition, two counterpart groups of noncryopreserved animals were bred and housed simultaneously to F1 and F2 generations (CF1 and CF2, respectively). The reproductive traits studied in all studied groups were litter size (LS), number born alive at birth (BA), and postnatal survival at Day 28 (number of weaned/number born alive expressed as percentage). The reproductive traits were analyzed using Bayesian methodology. Features of the estimated marginal posterior distributions of the differences between F1 and their counterparts (F1 − CF1) and between F2 and their counterparts (F2 − CF2) in reproductive characters found that vitrification and transfer procedures cause a consistent increase in LS and BA between F1 and CF1 females (more than 1.4 kits in LS and more than 1.3 BA) and also between F2 and CF2 females (0.96 kits in LS and 0.94 BA). We concluded that embryo cryopreservation and transfer procedures have long-term effects on derived female reproduction (F1 females) and transgenerational effects on female F1 offspring (F2 females).     

Complement Fixation Reaction for the Diagnosis of Type II Avian (Marek''s) Leukosis

The present study was designed to find a complement fixation (CF) reaction for the diagnosis of type II lymphoid leukosis, to learn some of the characteristics of the CF antigen, and to investigate the development of CF antibody response to this infection. JM virus-specific antigen was demonstrated in tumorous chicken tissue, in JM virus-infected chick embryo material, in JM virus-infected chicken kidney, and in duck embryo fibroblast tissue culture by using JM virus-immune rabbit serum. This CF antigen did not show cross-reactivity with Rous sarcoma virus or with RIF-type viruses. It was partially heat-labile. The CF activity was restored at -70 C for 10 months and was resistant to intermittent freeze-thaw treatment. The CF antigen may be denatured by ethyl alcohol, but no significant deleterious effects were noted after ether or chloroform treatment. JM virus-specific CF antibody could not be demonstrated by the direct complement dilution method or by the indirect or inhibition form of the CF test in infected or immunized chicken sera.     

Complement Fixation Reaction for the Diagnosis of Type II Avian (Marek's) Leukosis

The present study was designed to find a complement fixation (CF) reaction for the diagnosis of type II lymphoid leukosis, to learn some of the characteristics of the CF antigen, and to investigate the development of CF antibody response to this infection. JM virus-specific antigen was demonstrated in tumorous chicken tissue, in JM virus-infected chick embryo material, in JM virus-infected chicken kidney, and in duck embryo fibroblast tissue culture by using JM virus-immune rabbit serum. This CF antigen did not show cross-reactivity with Rous sarcoma virus or with RIF-type viruses. It was partially heat-labile. The CF activity was restored at —70 C for 10 months and was resistant to intermittent freeze-thaw treatment. The CF antigen may be denatured by ethyl alcohol, but no significant deleterious effects were noted after ether or chloroform treatment. JM virus-specific CF antibody could not be demonstrated by the direct complement dilution method or by the indirect or inhibition form of the CF test in infected or immunized chicken sera.     

The influence of culture vessel head-space volatiles on somatic embryo maturation in Sitka spruce [Picea sitchensis (Bong.) Carr.]

The maturation of somatic embryos of Sitka spruce [Picea sitchensis (Bong.) Carr.] was found to be highly dependent on the method used to seal plastic Petri dishes. Large numbers of well-formed mature embryos developed if dishes were sealed with PVC cling-film (CF) whilst sealing with Parafilm M (PF) greatly reduced the numbers of embryos forming. Inclusion of potassium permanganate oxidation traps, normally used to deplete the atmospheric ethylene, greatly stimulated somatic embryo maturation under PF sealing. Similarly, traps of adsorption agents (Tenax, activated charcoal or soft white paraffin), capable of removing volatiles from the culture vessel head-space, stimulated somatic embryo maturation under PF sealing although to a lesser extent than the oxidation traps. Incorporation of silver nitrate or 2-chloroethylphosphonic acid (ethephon) in the culture medium indicated that ethylene was not the agent supressing somatic embryo maturation under PF sealing.Abbreviations ABA abscisic acid - CF PVC cling-film - PF Parafilm M     

The osmotic property and fluorescent tracer movement of developing orchid embryos of Phaius tankervilliae (Aiton) Bl

The suspensor plays an active role during the early embryo development of flowering plants. In orchids, the suspensor cells are highly vacuolated without structural specializations, and the possible mechanism(s) that enable the suspensor to serve as the nutrient uptake site is virtually unknown. Here, we used the fluorescent tracer CFDA to characterize the pathway for symplastic transport in the suspensor cells of developing embryos and to provide direct visual evidence that the orchid suspensor has unique physiological properties. The embryo proper uptakes the fluorescent dye through the suspensor. CF could first be detected throughout the suspensor cell and then subsequently in the embryo proper. A plasmolysis experiment clearly indicates that suspensor cells have a more negative osmotic potential than the adjoining testa cells. It is proposed that the preferential entry of CFDA into the suspensor cell of the Nun orchid is aided by the more negative osmotic potential of the suspensor than neighboring cells, providing a driving force for the uptake of water from the apoplast into the symplast.     

Comparison of microinjection (piezo-electric) and cell fusion for nuclear transfer success with different cell types in cattle

Amongst the many variables that can determine success of cloning, the source of nuclei, the procedure used for nuclear transfer, and the activation of the reconstructed embryo are very important aspects. In this study, we have compared the two most common procedures for transferring nuclei to enucleated oocytes--cell fusion (CF) and piezoelectric microinjection (PEM) using different somatic cells--and we have investigated the effect of different activation procedures. Granulosa cells and fibroblasts were grown to confluency or in low serum to induce a quiescent state, while lymphocytes were thawed immediately prior to use. Enucleated oocytes were reconstructed either with CF or PME by 21-23 h postmaturation. For cell fusion, one pulse of 1 kVolt/cm for 30 microsec was used; for PEM, the cell membrane was broken by repeated pipetting and transferred in a 12% PVP solution to facilitate injection. Manipulated oocytes were activated with ionomycin and cycloheximide (CHX) or 6-DMAP (DMAP) and cultured in microdrops of SOF-BSA-AA. On day 7 (day 0: nuclear transfer), embryo development was evaluated and embryos were either transferred fresh or were frozen. More embryos were successfully reconstructed with PEM than CF, but a higher number of reconstructed embryos by CF developed to blastocyst at D + 7. In addition, in both systems more embryos were obtained after activation with DMAP than with CHX. The transfer of 141 embryos to recipients resulted in a pregnancy rate of 50%, and no differences were observed between the source of donor cell, the reconstruction methods, or the activation protocol. Six calves were delivered at term, and four survived. High pregnancy losses were observed throughout the gestation period.     

Embryo developmental disruption during organogenesis produced by CF-1 murine periconceptional alcohol consumption

The aim was to study the control females (CF)-1 mouse embryo differentiation, growth, morphology on embryonic E- and N-cadherin expression at midgestation after periconceptional moderate alcohol ingestion. Adult female mice were exposed to 10% ethanol in drinking water for 17 days previous to and up to day 10 of gestation (ethanol-exposed females, EF) and were compared with nonexposed CF. EF presented reduced quantities of E10 to E10.5 embryos, greater percentage of embryos at stages less than E7.5, reduced implantation site numbers/female, and increased resorptions compared with CF. EF-embryo growth was significantly affected as evidenced by reduced cephalic and body sizes of E10 and E10.5 embryos (scanning electron microscopy) and decreased protein content of E10.5 embryos vs. CF embryos. A significantly higher percentage of EF-E10-10.5 embryos presented abnormal neural tube (NT) closure vs. the percentage of CF. E10 embryos from EF presented elevated tissue disorganization, pyknosis and nuclear condensation in somites, mesenchymal and neuroepithelial tissue. Immunohistochemical E- and N-cadherin distribution patterns were similar in organic structures of E10 embryos between groups. However, western blot revealed that E- and N-cadherin expression levels were significantly increased in EF-derived embryos vs. controls. Perigestational ethanol consumption by CF-1 mice induced significant damage in the organogenic embryogenesis by producing delayed differentiation, growth deficiencies, and increasing the frequency of NT defects. Ethanol exposure may disrupt cell-cell adhesion leading to upregulation of E- and N-cadherin expression suggesting that deregulation of cell adhesion molecules could be involved in the disruption of embryo development at organogenesis in CF-1 mouse.     

Mouse zygote development in culture medium without protein in the presence of ethylenediaminetetraacetic acid

Development of zygotes from a hybrid-inbred (B6D2F1) and two random-bred (CD1 and CF1) strains of mice were compared after culture in several modifications of a simple, chemically defined medium based on Earle's Balanced Salts Solution. When cultured without the addition of protein or the chelating agent, ethylenediaminetetraacetic acid (EDTA), none of the zygotes reached the blastocyst stage. The addition of EDTA or protein significantly improved embryo development to blastocysts (p less than 0.05). The degree of improvement was dependent upon the strain of the female (85% or 91% for B6D2F1, 56% or 45% for CD1, and 19% or 28% for CF1, respectively). The addition of protein to the media in the presence of EDTA did not further improve embryo development. In all supportive conditions, zygotes from B6D2F1 females developed to blastocysts better than those from CD1 or CF1 females; embryos of the latter strain exhibited the lowest rates of development in vitro. Glycine and alanine (20 microM) partially substituted for EDTA; the decreased hybrid-inbred embryo development to blastocysts (20% and 26%, respectively) obtained in the presence of the amino acids suggested, however, that the stimulatory effect of EDTA on embryo development was other than as a source of fixed nitrogen. The rates of development observed with an alternate chelating agent, citric acid (less than or equal to 20% vs. 83% blastocysts, p less than 0.01), although better than the unsupplemented medium, were significantly less effective than EDTA-supplemented medium (83% blastocysts, p less than 0.01). The results of this study suggest that the protective effect of proteins in culture medium may be more important than their nutritive role.(ABSTRACT TRUNCATED AT 250 WORDS)     

Overcoming the 2-cell block by modifying standard components in a mouse embryo culture medium.

The 2-cell block may be caused by inappropriate concentrations of commonly used constituents of embryo culture media. Almost all zygotes obtained by fertilizing CF1 ova with hybrid B6D2F1/CrlBR sperm did not develop beyond the 2-cell stage when cultured in Whittingham's medium M16. This 2-cell block was overcome by lowering the concentrations of NaCl, KCl, KH2PO4, glucose, and pyruvate, either individually or in combination. The effects of changing the concentration of either NaCl or KCl depend on the concentration of NaHCO3 in the medium. Although a high percentage of embryos grew to the 4-cell stage in several media with lowered concentrations of certain components, the media are not optimal for complete preimplantation embryo development since the yield of blastocysts is low.     

Development of mouse embryos in vitro is affected by strain and culture medium

One-cell and two-cell embryos from three random-bred strains of mice–CF1, Dub:(ICR), and CFW (Swiss-Webster)–were cultured to the blastocyst stage in Spindle's, Earle's, Ham's F10, Whittingham's T6, or Hoppe and Pitts' medium. CFW embryos were more successful than CF1 and Dub:(ICR) embryos in developing to the blastocyst stage in all five media. Dub:(ICR) and CFW two-cell embryos showed the best development in Spindle's, Whittingham's T6, and Hoppe and Pitts', whereas CF1 two-cell embryos were most successful in developing in Hoppe and Pitts' medium. Similar results were obtained with one-cell embryos, although fewer developed to the blastocyst stage, and T6 rather than Hoppe and Pitts' medium sustained the best development of CF1 one-cell embryos. For all strains, the least successful development was in Ham's F10, but CFW embryos did show good development in this medium. In addition to the effects of various media on mouse embryo development, our results indicate that the strain of mouse used for the bioassay of media is of critical importance. Random-bred CFW (Swiss-Webster) mice are as suitable as a hybrid strain for this purpose.     

The role of Haemophilus somnus in bovine early embryonic death. III. The effect of the organism on embryos by day 21 postbreeding

A study using 11 healthy, mature Holstein-Friesian heifers was designed to determine whether 1) H. somnus induces gross and/or histopathological changes of the uterine tract and embryos, 2) H. somnus has a short and/or long-term effect on the ovarian activity, 3) prior exposure to H. somnus would modulate the effect of a second exposure to the organism. Superovulated heifers were artificially iseminated 12 and 24 h after standing estrus using high-quality, Haemophilus-free semen from a single ejaculate of one bull. Control heifers (Group 1, n = 2) were infused by intrauterine route, 12 h after the second insemination, with 10 ml of 0.85% sterile phosphate buffered saline (PBS) as a placebo. The treatment heifers were exposed by intrauterine infusion, 12 h after the second insemination, to approximately 1.5 x 10(9)H. somnus organisms (Iowa strain 1229) suspended in 10 ml of 0.85% sterile PBS. Group 2 (n = 2) treatment heifers were exposed 21 d prior to embryo recovery; Group 3 treatment heifers (n = 3) were exposed 5 mo prior to embryo collection; and Group 4 treatment heifers were exposed twice, 5 mo apart with the second exposure 21 d prior to embryo recovery. All animals were slaughtered and the whole genital tract was removed for pathological examination and embryo recovery. There were minimal pathological changes in the uterus. However, H. somnus significantly affected (P 相似文献   

Cystic fibrosis as a cause of infertility

Cystic fibrosis (CF) is one of the autosomal recessive diseases, caused by mutations in a gene known as cystic fibrosis transmembrane regulator (CFTR). The majority of adult males with CF (99%) is characterized by congenital bilateral absence of vas deferens (CBAVD). CBAVD is encountered in 1-2% of infertile males without CF. Females with CF are found to be less fertile than normal healthy women. In females with CF, delayed puberty and amenorrhoea are common due to malnutrition. CFTR mutations are also associated with congenital absence of the uterus and vagina (CAUV). The National Institutes of Health recommend genetic counseling for any couple seeking assisted reproductive techniques with a CF male or obstructive azoospermia which is positive for a CF mutation.     

Cleaning of Sendai virus-infected mice by embryo transfer technique

Mouse embryos (8-16 cells) were collected from Sendai virus (SV)-infected mice at 5 or 7 weeks after inoculation. All donors having embryo(s) were positive when tested by CF test or ELISA for SV antibody at the time of embryo collection. Most of the morphologically normal embryos developed (90.6%, 259/286) to morulae or blastocysts after culture for 26-28 hr. A total of 76 embryos cultured were transferred to the uteri of SV-free pseudopregnant recipients. Forty-seven young were obtained from these recipients (61.8% of development rate) and 46 young were successfully reared up to 10th week of age. All the recipients and the young were negative by testing SV antibody. These results indicate that the embryo transfer technique is useful for cleaning of SV-infected mice.     

Early Events in Development of Streptococcal Competence

Appropriately timed use of trypsin, which inactivates competence factor (CF), and chloramphenicol made feasible a separation and characterization of early events in the development of competence in group H streptococci. Step 1 is production of CF, which is inseparable in time from the concomitant release of free CF into the medium. The producing cells, which are noncompetent at the time, also accumulate cell-bound CF (CB-CF) from the onset of CF synthesis. In step 2, the released CF is adsorbed or taken up in a trypsin-insensitive state by the producing cells and is not destroyed as previously suggested. This occurs rapidly in a transformation-supporting (complete) medium. The rapid decline in free CF is concomitant with the rise in CB-CF, and a maximal increase in the latter does not occur in cultures exposed to trypsin, which inactivates any trypsin-accessible CF. The rapid increase in CB-CF (above trypsin-treated levels) leads to step 3, the induction of competence. All of these steps probably require protein synthesis, because each is inhibited by chloramphenicol. The data also indicate that only free CF that is subsequently adsorbed, and which thus leads to maximal levels of trypsin-insensitive CB-CF, is the effective inducer of competence in either CF-producing (Challis) or CF-nonproducing (Wicky) cultures. The processes induced by the newly bound CF are not fully understood, but certain new properties, previously described by others as indicating competence, were measured during the several steps of competence development. Cell aggregation at pH 2 appears to be related to CB-CF and can be shown before this bound CF has induced competence. The ability of cultures to autolyze maximally can be diminished by trypsin treatment of precompetent cells without affecting subsequent competence development as measured by transformation.     

Molecular screening of partners of cystic fibrosis heterozygotes.

We have screened 100 partners of known or suspected CF heterozygotes for ten CF mutations which account for 88% of the CF mutations seen by us on CF chromosomes. We have identified six CF heterozygotes amongst the 100 low-risk people screened. As two of the six people at high-risk of being CF carriers were subsequently shown not to be CF carriers this gave rise to four couples with a one in four risk of CF in a pregnancy and so far to two PND's. The risk of CF in the remaining 94 couples was reduced to less than one in 800. We have concentrated on the screening of partners for the commoner CF mutations rather than haplotyping them for the CF linked markers XV-2c/TaqI and KM19/PstI which are in linkage disequilibrium with CF. For individuals shown not to carry these ten mutations, a five fold difference in risk of being a CF carrier remains between those who have the XV-2c/KM19 genotypes associated with the highest risk(BB), as compared to those with the lowest risk(CC). Nevertheless we feel that effort is better expended in mutation screening rather than haplotyping.     

Subunit interactions within the chloroplast ATP synthase (CF0-CF1) as deduced by specific depletion of CF0 polypeptides

The proton-linked ATP synthase (CF1-CF0) of chloroplasts consists of a catalytic component (CF1) and a membrane-embedded part (CF0) that interacts with CF1 and contains a proton channel. The subunits of CF0 which are involved in binding of CF1 were studied by examining the effect of selective depletion of subunits I, II, and IV of CF0 from the chloroplast ATP synthase on the association of the remaining CF0 subunits with CF1. Dissociated CF0 subunits were identified by sucrose density gradient centrifugation. Removal of subunit IV alone from CF0-CF1 did not cause dissociation of the other CF0 subunits from CF1. Upon removal of both subunits I and IV from CF0-CF1, subunit II also dissociated, but subunit III was still bound to CF1. Thus, at least two subunits of CF0, I and III, directly associate with CF1. Subunit II is unlikely to bind CF1 directly and may associate with subunit I. Although depletion of subunit IV does not cause dissociation of CF0 from CF1, its interaction with CF1 subunits is uncertain.