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At least 127 records · Page 7Linked to original sources
PubMed1994

Generation of identical twin piglets following bisection of embryos at the morula and blastocyst stage.

Porcine morulae and blastocysts were microsurgically bisected and the resulting zona pellucida-free demi-embryos were either cultured in vitro for 48 h or transferred after 24 h of culture into--24 h asynchronous recipients. All demi-embryos were evaluated according to morphological criteria and classified into three categories (excellent, fair or degenerated). The average diameter and the number of cells were determined. Of 1162 bisected embryos, 764 pairs (66%) were evaluated as transferable after 24 h of culture in vitro. The average diameter after 48 h of culture in vitro was different (P < 0.01) among demi-embryos of the three morphological categories as was the number of cells. The greatest diameter and the greatest number of cells were found in demi-embryos classified as morphologically excellent. A total of 22 of 27 recipients (81.5%) remained pregnant and 21 recipients delivered 126 piglets of which six were stillborn. The survival rate of demi-embryos in farrowing recipients was 21.2% (126 of 594). Litter size was significantly reduced in recipients after transfer of demi-embryos compared with that of mated controls (6.0 +/- 2.5 versus 10.8 +/- 2.1 piglets). Similarly, the postpartum losses of piglets were higher in the experimental than in the control gilts (26.7% versus 11.6%). Duration of gestation, average birth weight and daily weight gain were not affected. Among the 126 piglets, seven pairs of identical twins (2.3% of 311 transferred pairs) were identified using several genetic markers in blood (blood groups, polymorphic enzymes and plasma proteins) in a total of 25 gene loci. DNA fingerprinting revealed an identical banding pattern between the two partners of each of the seven pairs. Birth and weaning weight as well as daily weight gain varied considerably between monozygotic partners.

Animals↗
PubMed1992

Isolation of plasma membranes, Golgi bodies and mitochondria of Xenopus laevis morulae. Identification of plasma membrane proteins.

Homogenates of Xenopus morulae at the 16-32 cell stage were centrifuged on discontinuous sucrose gradients. Isolated fractions were identified by electron microscopy (EM) as mitochondria, a fraction enriched in Golgi vesicles, and plasma membranes. A special effort was made to prepare plasma membranes free of cytoplasmic contaminants. The resulting purified plasma membranes appeared morphologically identical to plasma membranes in situ. The external surface is covered with a fibrillar coat while vesicles are seen attached to their inner surface. O'Farell's method (1975) was used to obtain protein patterns of the various fractions on 2-dimensional gel electrophoresis. Each fraction displayed a specific pattern. By comparing the different patterns, it was possible to identify a group of proteins as belonging to the plasma membrane fractions. Labelling of cell surface with sulfo-N-hydroxysuccinimido-biotin together with differential extraction of proteins has allowed us to tentatively allocate these proteins in different structures of the plasma membrane fractions. The data presented in this paper corroborate and extend our ultrastructural studies on neogenesis of interblastomeric plasma membranes (Bieliavsky and Geuskens, 1990).

Animals↗
PubMed1991

Bisection of bovine morulae and blastocysts from superovulated Danish dairy cows.

Sixtyfour compacted morulae and blastocysts were bisected with a microscalpel. The majority of the demi-embryos (n = 122) were reinserted into separate zona pellucidae (ZP) before non-surgical transfer to 113 synchronized recipients, as singles (n = 98) (DE-S) or in pairs (n = 30) (DE-P). Thirty non-manipulated embryos (E) were transferred during the same period and served as controls. Pregnancies were diagnosed by rectal palpation 4-7 weeks after transfer. The pregnancy rates for DE-S, DE-P and E were 32%, 53% and 40%, respectively (P greater than 0.05). A substantial number of abortions were recorded between 50 and 250 days of pregnancy among the recipients with DE-S. The fetal survival rate for DE-S was reduced to 21% and significantly lower (p less than 0.05) than the survival rates of DE-P (43%) and E (40%). The quality of DE and the presence of ZP did not significantly influence the results. No conclusive reasons for the fetal loss could be found but different possibilities are discussed.

Animals↗
PubMed1990

The morula-blastocyst transition in two Old World primates: the baboon and rhesus monkey.

Baboon and rhesus monkey embryos demonstrate compaction during the morula stage, although not all blastomeres participate simultaneously. During this process the outer adherent cells develop progressively more extensive apical junctional complexes. Several spaces appear between blastomeres before formation of a single cavity, and assortment of inner cell mass and trophoblast cells is less rapid and less precise in primates than in rodents. Nonhuman primates should prove appropriate for studies of individual aberrant blastomeres during blastocyst formation.

Animals↗
PubMed1990

Ultrastructural studies on the morula in cattle.

Embryos at morula stage were ultrastructurally examined. Blastomeres of normal embryos are characterized by the presence of ribosomes, short single and branched cisterns of endoplasmic reticulum, small spherical mitochondria and vacuolar-granular structures. An enhanced autophagy and the presence of numerous and large lipid droplets are signs of the beginning degeneration.

Animals↗
PubMed1982

The possible mechanism of cell positioning in mouse morulae: an ultrastructural study.

Ultrastructure of the mouse morula at the time of formation of the outer cell layer is described. Analysis of relative positions of sister blastomeres, still connected with cytoplasmic bridge, suggests that cell division may occur both tangentially and perpendicularly in relation to the embryo surface. However, there are observations suggesting that active cell movement may be involved in cell positioning already after mitosis. These observations include: (1) the flexure of cytoplasmic bridge, (2) the presence of bundles of filaments in external blastomeres at the late cytokinesis and (3) a striking difference in loose versus tight packaging of blastomeres within the embryo.

Animals↗
PubMed1980

Distribution of antibody- and lectin-binding sites on dissociated blastomeres from mouse morulae: evidence for polarization at compaction.

The distribution of binding sites for rabbit anti-species antiserum, Concanavalin A (Con A) and peanut agglutinin (PNA) on dissociated blastomeres from 2- to 16-cell mouse embryos has been investigated using direct and indirect fluorescence techniques. With each ligand, paraformaldehyde-fixed blastomeres from 2- to 8-cell precompact embryos were uniformly surface labelled; the majority (77%) of late compact 8-cell blastomeres showed quantitative polarization of surface labelling; and 16-cell blastomeres were either polarized (53.3%) or uniformly surface labelled. Binding of fluorescein-conjugated PNA increased at the 16 cell stage. Labelling patterns on unfixed blastomeres were similar to those on fixed blastomeres except that surface label was patched and became internalized, most rapidly from the less heavily labelled areas of 8- and 16-cell blastomeres. Quantitative polarization of binding sites at postcompaction stages was detected after (i) fixation, (ii) pretreatment and labelling in the presence of azide, cytochalasin D and/or colcemid, or (iii) labelling with monovalent Fab1 antibody fragments. It is probably due, therefore, to the presence of microvilli at the heavily labelled pole, which increase surface area and are known to become to the outer surface of the compact morula (Ducibella, Ukena, Karnovsky & Anderson, 1977). The possibility that the cleavage of polarized blastomeres into dissimilar daughter blastomeres could provide a mechanism for the spatial differentiation of the inner cell mass and trophectoderm of the blastocyst is briefly discussed.

Animals↗
PubMed1993

Nuclear transfer in cattle: birth of cloned calves and estimation of blastomere totipotency in morulae used as a source of nuclei.

The birth of a clone of five bull calves, each produced by fusing a cell taken from the same young embryo with an oocyte whose own nucleus has been removed, opens the way to the use of these new animal models for research and animal selection. The embryonic cells came from a 31-cell morula. Our results indicate that most of the nuclei at this stage are totipotent which seems to no longer be the case for those taken from the cells of the inner cell mass at the blastocyst stage.

Animals↗
PubMed1999

Murine Pkd1 is a developmentally regulated gene from morula to adulthood: role in tissue condensation and patterning.

PKD1 is the most common genetically mutated gene involved in autosomal dominant polycystic kidney disease (ADPKD). Our previous studies have shown that the pathogenesis of human and murine polycystic kidney disease (PKD) involves failure to switch out of a renal developmental program, suggesting a role for PKD1 in development. To investigate this hypothesis, we have cloned a portion of the murine Pkd1 gene and characterized the fetal to adult tissue expression pattern of Pkd1. We chose to clone the transmembrane region of Pkd1, a region prone to mutations in ADPKD. The transmembrane coding region (2.6 kb) has 80.3% nucleotide homology with human PKD1 and 85.3% amino acid similarity. The cloned murine Pkd1 fragment closely resembles that of human PKD1 with respect to both genomic size and exon/intron position. We have demonstrated that this Pkd1 region is not conserved in lower organisms and is mammalian specific. A detailed expression analysis of Pkd1 revealed expression as early as the morula stage and in ES cells with differential expression levels in various tissues/organs throughout development. Highest expression levels were observed in the early condensing mesenchyme of primitive mesoderm and ectoderm. Pkd1 was also expressed at high levels in developing neural tube, neural crest derivatives, prechondrogenic tissue, metanephros, bladder, salivary glands, lung, and blood vessels with lower expression levels in other organs and tissues. Specific spatial and temporal patterns of Pkd1 expression were demonstrated in individual organs, such as lung, kidney, brain, indicating it is highly developmentally regulated. Particularly high levels persisted in mature derivatives of neural tube, neural crest, chondrogenic tissue, metanephros, and lung. In summary, our data suggest that Pkd1 has at least two cellular functions, one a basic function involved in early tissue condensation processes, and the other a mammalian-specific function, that evolved with tissue patterning and tubulogenesis in metanephric and pulmonary development.

Animals↗
PubMed1997

Morulin Pm: a modified polypeptide containing TOPA and 6-bromotryptophan from the morula cells of the ascidian, Phallusia mammillata.

A novel polypeptide containing the unusual posttranslationally modified amino acids L-3,4,5-trihydroxyphenylalanine (TOPA) and L-6-bromotryptophan (6-BrW) has been isolated from the morula cells of the vanadium-accumulating ascidian, Phallusia mammillata. The polypeptide, designated Morulin Pm, has a molecular weight of 3825 +/- 0.6 and has a simple amino acid composition consisting mainly of TOPA and 6-BrW as well as Ser, Leu, Phe, and Ala. To our knowledge, this is the first reported example of multiple sites of brominated tryptophan in a polypeptide of this size. Edman degradation revealed the N-terminal sequence to be BrW-Leu-Phe-BrW before sequencing was blocked. While the N-terminal tripeptide could be isolated from chymotrypsin digests of Morulin Pm, the rest of the polypeptide resisted further cleavage by the proteases, a feature common among this class of peptides. However, unlike other ascidian blood cell peptides examined to date, microheterogeneity was minimal. For the first time a detailed NMR investigation could be undertaken on a member of this class of polypeptides. In addition to signals assignable to the constituent amino acids by extensive 2D experiments, resonances were present both in the 13C and 1H spectra not typical of a simple linear peptide. Two proton resonances were identified with a cross peak in the correlation spectrum strongly indicative of a C-terminal decarboxy-delta 2,3-unsaturated TOPA residue as observed in certain tunichromes and clionamide. Chemical degradation experiments were undertaken in an effort to produce identifiable fragments to which these signals could be assigned, including full and partial acid hydrolysis and tryptophan-targeted BNPS-skatole treatment. However, the nature of the modification remains unknown. Possible structures for the modification, which may represent the source of the difficulties encountered in the structural elucidation of this and related peptides, are assessed. Conjecture is made as to the biological relevance of Morulin Pm, based on its localization and chemical characteristics.

Amino Acids↗
PubMed2006

Mutational analysis of the APC gene in cribriform-morula variant of papillary thyroid carcinoma.

INTRODUCTION: Familial adenomatous polyposis (FAP) is an inherited autosomal dominant syndrome caused by germline mutations in the adenomatous polyposis coli (APC) gene. Papillary thyroid cancer is one of the extracolonic manifestations of FAP. A characteristic histologic feature of this type of thyroid tumor is the cribriform-morula variant of papillary thyroid carcinoma (CMVPTC). METHODS: To investigate roles of the APC and beta-catenin genes in the development of CMVPTC, we examined germline and somatic mutations of these genes in a female patient with CMVPTC and FAP. The patient had undergone total colectomy at the age of 19 years and total thyroidectomy at age 25 years. RESULTS: Numerous tumors were disseminated in both lobes of the thyroid gland, and histopathologic examination revealed typical CMVPTC. DNA was extracted from peripheral blood leukocytes and 12 CMVPTC tumors, and exons 1-15 of the APC gene and exon 3 of the beta-catenin gene were examined. A germline mutation was detected in exon 13 of the APC gene, and this mutation generated a premature stop codon. Six somatic mutations (922delC, 1602delA, 1821delT, 1920delG, 2706del20, 2804insA) were found in the CMVPTC specimens. All mutations were truncating mutations in the N-terminus of the APC protein. Loss of heterozygosity was not observed in the remaining tumor tissues without somatic APC mutations. There were no mutations of the beta-catenin gene in peripheral blood leukocytes or 12 CMVPTC specimens. CONCLUSIONS: These results suggest that APC mutations play an important role in the development of CMVPTC and occur predominantly in the 5' side of the APC gene between codons 308 and 935.

Adenocarcinoma, Papillary↗
PubMed1985

Polarity of isolated blastomeres from mouse morulae: detection of transcellular ion currents.

Eight- to sixteen-cell stage mouse morulae were dissociated with Ca2+-free medium into blastomeres that were labeled with fluoresceinated-succinylated Con A (FS-Con A) to mark their apical-basal axes. The vibrating probe was then used to map their extracellular current patterns. The average current density around normal blastomeres approached the resolution of the probe system (0.2 microA/cm2) and was undetectable in the majority of blastomeres. Since the current density at the measuring point outside the cell is known to increase with cell size in other systems, enlarged blastomeres were created by fusing together blastomeres of 4-cell stage embryos in 45% polyethylene glycol. Enlarged blastomeres were then aggregated with normal blastomeres using phytohemagglutinin and cultured to the 8- to 16-cell stage to allow them to become polarized. Such aggregates were then dissociated with Ca2+-free medium to recover polarized, enlarged blastomeres. The enlarged blastomeres were 30-65 microns in diameter and 70% of them generated a detectable current; currents were detected around 83% of those blastomeres larger than 40 micron in diameter. The current pattern in these most reliable cases was predominantly inward apical (11/16 or 69%) and outward basal (15/16 or 94%), with lateral currents about three-fold smaller in amplitude than these apical-basal currents. Lateral currents were undetectable in 53% of the cases. Preliminary data suggest that the inward current is carried in part by Na+ influx and is independent of the Na+,K+-ATPase over the short term. Transcellular ion currents were detectable as long as 4 hr after dissociation, and the apical-basal current pattern was usually stable during that time. In contrast, the fluorescent cap of FS-Con A faded within 7-30 min at 35 degrees C but remained stable in 0.1% azide or 1.5 micrograms/ml cytochalasin D. The electrical polarity therefore persisted after the apical cap of Con A fluorescence was no longer visible. We propose that these transcellular ion currents may be involved in the establishment of blastomere polarity and describe a mechanism of action in an "ion current polarization" hypothesis.

Animals↗
PubMed1981

Chromosome preparation from single blastomeres after colcemid treatment and removal from rabbit morulae -- unsuitable for sexing in routine embryo transfer.

Cell cycle of blastomeres of 3 days old rabbit morulae were synchronized by exposing the entire embryo to colcemid in BSM-culture-medium. Few cells were removed by micropuncture and prepared for chromosome analysis. Neither colcemid treatment nor removal of cells appears to harm the embryo. Successful diagnosis of embryonic sex was performed in 14% of all embryos only. The poor success is due to a mitotic rate of less than 10 %. The method appears unsuitable for routine use.

Journal Article↗
PubMed1983

Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves.

Mature Brangus donor cows were superovulated with follicle stim-ulating hormone administered twice daily in intramuscular injections. On day 6.5 to 7 post-estrus, embryos were collected non-surgically using a phosphate-buffered saline medium. A total of 37 ova was collected, of which 28 were advanced morulae and early blastocysts. Twenty of these embryos were selected for micromanipulation with a radial-type Leitz micromanipulator. While the embryos were in a holding medium containing 10% fetal calf serum, three glass microinstruments were used to open the zona pellucida, remove the mass of blastomeres and bisect the embryo on a vertical plane. Halved embryos were inserted into bovine zonae and placed either as single half-embryos or twin half-embryos in 0.25 ml French straws with fresh holding medium. The micromanipulated embryos (demi-embryos) were then non-surgically transplanted, either as a single demi-embryo or as a twin demi-embryo pair, into the uterine horn of day 6.5 to 8 recipient beef females ipsilateral to the existing corpus luteum. Of the 14 micromanipulated embryos that were transplanted to recipients, pregnancy rates were 16.6% for the single demi-embryos and 62.5% for the twin demi-embryos. No pregnancies resulted from bisected blastocysts.

Journal Article↗
PubMed1984

Chimerism in cattle through microsurgical aggregation of morulae.

A cattle chimera was produced by combining four halves of two parent embryos of different breeds (Brown-Swiss x Braunvieh plus Holstein-Friesian x Holstein-Friesian) in one zona pellucida. Parent embryos in the 32-cell morula stage were recovered non-surgically, were bisected, and the combined four halves were transferred non-surgically to recipient heifers. Chimerism of coat colour was used as evidence. Combining of only two half embryos from different parents resulted in five pregnancies carried to term but none of the calves born was a chimera.

Journal Article↗
PubMed1984

Survival rate to term of chimeric morulae produced by aggregation of five to nine embryos in the mouse, Mus musculus.

Chimeric morulae from five to nine embryos were produced by aggregation after removal of the zonae pellucidae and brief incubation in phytohemagglutinin-P. After 28 hr of in vitro culture, these aggregates were transferred to the left uterine horn of pseudopregnant recipient female mice. Each recipient also received control embryos in the right uterine horn. Genetic markers were included so that aggregate-derived offspring could be distinguished from control offspring. Aggregate-derived embryos survived to term, but at a much reduced rate. In the 25 recipients that produced litters, 6.2% (N=211) of the aggregated embryos developed into liveborn young. Survival-to-term for control embryos was 38.8% (N=387). Survival-to-term was independent of the number of embryos aggregated. The aggregate-derived embryos were apparently undergoing size regulation following implantation.

Journal Article↗
PubMed1985

Observations on in vitro development of bovine morulae in Ham's F-10 and Dulbecco's phosphate buffered saline supplemented with normal steer serum.

This study was conducted to compare in vitro development of bovine morulae in Ham's F-10 and Dulbecco's phosphate buffered saline (D-PBS) media supplemented with 10% (v/v) normal steer serum. Fifty-three excellent and good embryos were obtained by superovulating 15 non-lactating Holstein cows. Embryos were placed randomly in culture with Ham's F-10 or D-PBS media and development was recorded at 12-h intervals for the duration of culture. All embryos reached early blastocyst, blastocyst and expanded blastocyst stage. Nineteen of 27 embryos (70.1%) cultured in Ham's F-10 developed to hatched blastocyst stage in contrast to three out of 26 in D-PBS (11.5%). The mean developmental scores at 24, 48, 72, 96 and 120 h of culture were significantly (P<0.001) higher for embryos cultured in Ham's F-10. Also, the mean times to reach early blastocyst (25.84 +/- 6.65 vs 46.67 +/- 9.99 h), blastocyst (44.57 +/- 11.45 vs 61.89 +/- 16.62 h) and expanded blastocyst stage (65.00 +/- 13.20 vs 73.41 +/- 15.80 h) were significantly (P<0.001) shorter for embryos cultured in Ham's F-10. No difference was observed in the mean time to reach hatching (90.00 +/- 10.85 vs 84.00 +/- 16.97 h) and hatched blastocyst stage (97.26 +/- 18.71 vs 96.00 +/- 0.00 h). The results obtained support the concept that Ham's F-10 and normal steer serum provide for optimal bovine embryo development and suggest that 10% normal steer serum could be used as a protein supplement with D-PBS for short term storage and culture of bovine embryos.

Journal Article↗
PubMed1986

Effect of silver iodide as an ice inducer on viability of frozen-thawed rabbit morulae.

A new method was devised for inducing ice crystal formation in extracellular solution using silver iodide. A latent heat occurred immediately before temperature of sample reached -7 degrees C, when a column 70 mm high of 1.5M dimethyl sulfoxide (the freezing solution, FS) was aspirated into a plastic straw followed by 3 mm high of air and 10 mm high of 1% suspension of silver iodide in distilled water (1% AgI). To examine the effect of silver iodide as an inducer of ice crystal formation in extracellular solution on in vitro development of frozen-thawed rabbit morulae, the straws were filled by successive aspiration of the following fractions: 175 mul of FS containing the embryos, 7.5 mul of air, 25 mul of 1% AgI. The straws were cooled to -7 degrees C at 1 degrees C/min, and held at -7 degrees C for 10 min without initiating seeding; they were then cooled again to -30 degrees C at 1 degrees C/min and plunged into liquid nitrogen. After rapid thawing (>1000 degrees C/min), 100 of 109 (92%) embryos that were recovered developed into expanding blastocysts.

Journal Article↗