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PubMed1993

In vitro and in vivo development of mouse morulae encapsulated in 2% sodium alginate or 0.1% poly-l-lysine.

In Experiment 1, development of zona pellucida-intact (ZPI) morulae was measured every 24 hours for 120 hours after encapsulation in 2% sodium alginate (ALG) or 0.1% poly-L-lysine (PLL). Encapsulation significantly reduced development to hatched blastocysts at 48 and 72 hours. Developmental stages and diameters of ZPI and zona pellucida-free (ZPF) unencapsulated and encapsulated morulae were measured every 24 hours for 72 hours in Exeriment 2. At 72 hours, the percentage of ZPI embryos developing to expanded blastocysts, their diameters and their nuclear counts were not different from each other or from ZPF embryos. In Experiment 3, ZPI morulae encapsulated in ALG or PLL were transferred into recipients. Five of six recipients that received unencapsulated embryos (n=71) delivered 16 live pups. None of the recipients of encapsulated embryos delivered offspring; therefore, a final experiment was performed to examine fetal development on Day 10 of gestation. The percentage of pregnant recipients was similar for all 3 treatments: unencapsulated (71.4%), ALG (87.5%) and PLL (87.5%). However, the presence of viable fetuses was higher for unencapsulated embryos (42.1%) than for ALG (17%) and PLL (14.6%) embryos. These results suggest that encapsulation did not detrimentally affect embryonic size or cellular development in vitro; however, mortality occurred in vivo due to an asynchronous condition between the uterine environment and the embryos.

Journal Article↗
PubMed1990

The intracellular pH of tunicate blood cells: Ascidia ceratodes whole blood, morula cells, vacuoles and cytoplasm.

The intracellular pH of blood cells of the tunicate Ascidia ceratodes has been measured by equilibration of radioactively labeled markers between intra- and extracellular media. Labeled acid, 5,5-dimethyloxazolidine-2,4-dione (DMO), and base, methylamine (MA), have been used in the range of extracellular pH (pHm) of 4.5-7. For unsorted blood cells MA is less sensitive to the transmembrane pH gradient (delta pH) than is DMO in the pHm of 6.3-7. The data measured by DMO yield an intracellular pH value of 6.98 +/- 0.15. Ficoll density gradients separated 86.4% pure morula cells. Other experiments show that morula cells contain significant amounts of vanadium and most of the free tunichrome. Using both MA and DMO with morula cells yields pH values of 5.0 +/- 0.2 for the vacuoles and 7.1 +/- 0.2 for the cytoplasm. If vanadium is accumulated in the intravacuolar solution space, then this mildly acidic pH indicates that the aquo V3+ ion, which is only stable below pH 3, is stabilized by some factor other than high hydrogen ion concentration. This factor may be chelation by tunichrome. It is also possible that accumulated vanadium(III) is sequestered in hydrophobic regions of the vacuolar or cellular membranes.

Animals↗
PubMed2004

A global view of gene expression in the preimplantation mouse embryo: morula versus blastocyst.

As a first step to understand preimplantation development, we performed global gene-expression profiling of morula and blastocyst using the NIA 15k mouse cDNA microarray. Gene expression levels were measured four times for blastocyst and five times for morula. Student's t-test at the 5% significance level identified 428 genes upregulated and 748 downregulated in blastocyst compared to morula. This trend was consistent with semi-quantative RT-PCR analysis of sample genes. The upregulated genes known to be involved in critical regulatory processes, included Mist1, Id2, Hd1, and Requiem; the downregulated genes included CREB-binding protein, Per3, zinc finger protein 217, Krox-25, and miwi1. Such well-characterized genes and many novel genes provide markers for early stages in development and starting materials for further functional studies.

Animals↗
PubMed1978

Demonstration of a chorionic gonadotropin-like substance in rabbit morulae.

Rabbit morulae were treated with specific indirect immunofluorescence and immunoperoxidase histochemical techniques. The first antibody in both systems was rabbit anti-human chorionic gonadotropin (hCG) beta-subunit. Negative controls revealed a complete absence of reaction. All morulae incubated with the double-antibody system showed a positive reaction. Rabbit morulae thus present a substance with antigenic determinants similar to the beta-subunit of hCG. The physiologic role of this substance is unknown.

Animals↗
PubMed2001

Glucose metabolism of human morula and blastocyst-stage embryos and its relationship to viability after transfer.

The pregnancy rate and implantation rate following blastocyst transfer in the human have been reported to be high; however, it has remained necessary to transfer 2-3 blastocysts to achieve these rates. Morphological criteria are currently used to select blastocysts for transfer and have some limited correlation with ongoing viability. Glucose metabolism of 189 human morula to blastocyst stage embryos was analysed using a non-invasive ultramicrofluorescence technique to determine if this could be used to predict viability. There was a linear trend to increased glucose uptake with progression from the morula to the hatching/hatched blastocyst stage of development, whereas glycolytic activity did not vary. There was no consistent difference in glucose uptake or glycolytic activity for embryos at the various morphological stages on day 5 compared to day 6 in vitro. Glucose uptake and glycolytic activity of the nine embryos positively identified as having implanted following transfer varied and were apparently not different from the values for embryos that failed to implant. In addition, viability was demonstrated to be compatible with high glycolytic activity, with four of nine implanted embryos having a glycolytic activity in the highest 15% of the population of embryos studied. Glucose uptake and glycolytic activity of male and female embryos did not appear to be different. Glucose metabolism cannot be used prospectively to select viable human morula or blastocyst stage embryos for transfer and it is also unlikely to be a useful tool to predict the sex of the embryo.

Journal Article↗
PubMed2003

Cloning and characterization of an Ehrlichia canis gene encoding a protein localized to the morula membrane.

A gene encoding a 23.5-kDa ehrlichial morula membrane protein designated MmpA was cloned by screening an Ehrlichia canis expression library with convalescent dog sera, which resulted in three positive clones. Sequence analysis of the insert DNAs from all three clones indicated an open reading frame with a size of 666 bp that encodes MmpA. The structural analysis of MmpA indicated that it is a transmembrane protein with extreme hydrophobicity. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated the presence of a single copy of the mmpA gene in E. canis and Ehrlichia chaffeensis but not in the human granulocytic ehrlichiosis agent. The mmpA gene was amplified, cloned, and expressed as a fusion protein. Polyclonal antibodies to the recombinant protein (rMmpA) were raised in rabbits. Western blot analysis of E. canis and E. chaffeensis lysates with the anti-rMmpA serum resulted in the presence of an MmpA band only in E. canis, not in E. chaffeenesis. Sera from dogs which were either naturally or experimentally infected with E. canis recognized the recombinant protein. Double immunofluorescence confocal microscopy studies demonstrated that MmpA was localized mainly on the morula membrane of E. canis. Since the morula membrane is the interface between the ehrlichial growing environment and the host cytoplasm, MmpA may play a role in bacterium-host cell interactions.

Amino Acid Sequence↗
PubMed1992

Identification of Ehrlichia chaffeensis morulae in cerebrospinal fluid mononuclear cells.

We report a case of ehrlichiosis in a 72-year-old man who developed extreme lethargy, acute renal failure requiring hemodialysis, and respiratory insufficiency requiring intubation. Lumbar puncture performed on the second day of hospitalization revealed significant cellular pleocytosis. Ehrlichia morulae were tentatively identified in mononuclear cells in routinely processed Wright-stained cytospin preparations of cerebrospinal fluid (CSF). Identification was confirmed by a specific immunocytochemical staining procedure. Subsequent identification specifically as Ehrlichia chaffeensis morulae was established by polymerase chain reaction analysis, which revealed E. chaffeensis-specific DNA in CSF, bone marrow, and blood samples; by indirect fluorescent-antibody analysis, the patient developed an antibody titer of 32,768 against E. chaffeensis antigen. The patient responded to intravenous therapy with doxycycline and dexamethasone. Subsequently, neurologic, hematologic, renal, and pulmonary status had returned to baseline at follow-up 12 weeks after admission. To our knowledge, this is the first identification of E. chaffeensis morulae in CSF cells in an infected patient.

Aged↗
PubMed1976

Survival of 16-celled and morula stage rabbit embryos frozen to -196 degrees C,.

Preimplantation stage (16-celled and morula) rabbit embryos were successfully frozen to -196 degrees C. The cooling rate (from a room temperature to 0 degrees C), the presence of the mucin layer surrounding embryos, the ice-seeding treatment and the thawing procedure were examined to determine their effects on the survival of the frozen embryos of Japanese white, New Zealand white and Dutch-Belted rabbits. A high proportion (51%; 16-celled, 69%; morula) of Dutch-Belted rabbit embryos developed in vitro, when they were frozen to -196 degrees C, applying the ice-seeding at -4 degrees C in the presence of 12.5% DMSO, after being cooled to 0 degrees C at the rate of 7-9 degrees C/min, and were diluted by a stepwise addition of 4 different strength PBS on thawing. The highest rate of in vitro development (81%; Japanese white, 75%; New Zealand white, 82%; Dutch Belted embryos) was obtained when the morula stage embryos were frozen to -196 degrees C applying seeding at -4 degrees C after being cooled to 0 degrees C at the rate of 1 degrees C/2.5 min and were diluted, on thawing, by stepwise addition of 6, 3 and 1% DMSO solution and a culture medium. No great difference was found in the survival rate between the embryos covered with the mucin layer and those which had not the coat. All the embryos frozen without applying seeding treatment failed to develop in vitro after being thawed and diluted. Nine out of 27 does each of which received 6 reimplantations of the embryos frozen-thawed became pregnant and were found to be carrying 37 normal fetuses on the 12th day of pregnancy.

Animals↗
PubMed1975

Investigation of the determinative state of the mouse inner cell mass. I. Aggregation of isolated inner cell masses with morulae.

Inner cell masses (ICMs) were dissected from 3 1/2- and 4 1/2-day blastocysts and cultured in contact with 2 1/2-day morulae. Blastocysts and morulae were homozygous for different electrophoretic variants of the enzyme glucose phosphate isomerase (GPI). Aggregation of ICMs and morulae was observed, and such aggregates were able to form blastocysts in vitro and morphologically normal foetuses in utero. GPI analysis of these conceptuses revealed that most were chimaeric. However, donor ICM-type isozyme was only detected in the embryonic and extra-embryonic fractions of the chimaeras and never in the trophoblastic fraction. Thus, ICM cells appear unable to form trophoblast derivatives even when exposed to 'outside' conditions as experienced by developing trophoblast cells. This is evidence that ICM cells, although not overtly differentiated, are determined by 3 1/2 days.

Animals↗
PubMed2001

Assessment of metabolism of equine morulae and blastocysts.

Nutrient uptakes and metabolite production by equine morula and blastocyst stage embryos were determined by non-invasive microfluorometry. Equine morula took up equal amounts of both pyruvate and glucose. However, at the early blastocyst there was a small increase in glucose uptake and, by the expanded blastocyst stage, glucose was the predominant nutrient. Expanded blastocysts took up five times more glucose than pyruvate. Expanded blastocysts exhibited an exponential increase in glucose uptake and lactate production with respect to both diameter and surface area. As less than 50% of the glucose was accounted for by lactate production, the equine blastocyst appears to have a significant capacity to oxidize glucose. Embryos with a higher morphological grade consumed more nutrients than those with a poorer morphology. However, there was a large range in nutrient consumption within the highest grade blastocysts. This suggests that nutrient uptake may be useful as a viability marker of equine blastocysts.

Animals↗
PubMed1989

Microsurgical bisection of porcine morulae and blastocysts to produce monozygotic twin pregnancy.

Porcine morulae were bisected by a glass needle after softening of zonae pellucidae by pronase followed by a treatment with 0.05% trypsin/0.02% EDTA for decreasing intercellular junction of blastomeres. Transfer of 49 half-morulae to three recipients resulted in one pregnancy. The blastocysts were bisected symmetrically so as to leave a cellular bridge between the sister half-embryos after the softening of zonae followed by or without the trypsin/EDTA treatment. Transfer of 47 monozygotic (MZ) pairs of half-blastocysts to nine recipients resulted in four pregnancies. A litter of nine fetuses was obtained from seven MZ pairs of half-blastocysts, demonstrating that at least two pairs of MZ twin fetuses were produced. It is thought that the procedure for bisecting blastocysts developed in this study is one of the potential methods of producing porcine MZ twins.

Animals↗
PubMed2005

Impact of pro-oxidant agents on the morula-blastocyst transition in bovine embryos.

Exposing day 5 bovine morulae to reactive oxygen species induces a delayed degeneration of some blastocysts on day 8 post-insemination (pi) but without affecting the blastocyst rates. The aim of this study was to characterize the resisting and the degenerating population of blastocysts. The kinetics of degeneration of the embryos exposed to the two pro-oxidant agents: 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and buthionine sulfoximine (BSO) was evaluated using time-lapse cinematography. With both agents the first signs of degeneration appeared at day 7.5 pi but the duration of the degeneration process was shorter in presence of AAPH than BSO (4.2 vs. 12.5 hr, ANOVA, P < 0.05). The resisting blastocysts derived from morulae with a larger diameter (mean diameter: 161 vs. 154 microm, ANOVA, P < 0.05) and showed an earlier cavitation (135 vs. 142 hpi, P < 0.05) than the degenerating ones. The profile of protein neosynthesis at day 7 was not affected by the treatment. The proportion of male embryos was more important in the resisting than in the degenerating population (70 vs. 55%, chi2, P < 0.05) especially when the stress was induced by AAPH. The quality of the resisting embryos, measured by the total cell number and the rate of apoptosis, did not seem to be affected when compared to control embryos. In conclusion, resistance to oxidative stress seems related to the kinetics of development and/or the sex of the embryos. Resisting embryos apparently display a quality similar to untreated embryos.

Animals↗
PubMed1993

Morula decompaction (mdn), a preimplantation recessive lethal defect in a transgenic mouse line.

The beta S12 transgenic line carries an insertion of exogenous DNA and a deletion of approximately 2 cM on chromosome 1. Transgenic heterozygotes (beta S12/+) were viable and fertile, but homozygous mice (beta S12/beta S12) could not be produced. To determine the stage at which developmental arrest occurred in homozygotes, embryos derived from intercrosses among heterozygotes or from control crosses were examined at various stages. No homozygotes could be detected at postimplantation stages, suggesting that the mutation affected embryogenesis before implantation. Recovery of embryos at 1.5 or 3.5 days postcoitum, followed by culture in vitro, revealed an abnormal class of embryos which constituted one-fourth of the progeny from intercrosses, and thus appeared to represent the beta S12/beta S12 homozygotes. These embryos cleaved normally to the 8-cell stage and formed compacted morulae, but then decompacted without forming a blastocyst, and ceased dividing at approximately the 16-cell stage. Nearly all blastomeres were viable at the time of decompaction, as demonstrated by trypan blue exclusion, indicating that the loss of compaction is not simply a consequence of cell death. When labeled with a short-term lineage marker and aggregated with normal embryos at the early 8-cell stage, homozygous mutant embryos failed to contribute to the resulting blastocysts, indicating that the defect in the maintenance of compaction is cell autonomous. Based on the mutant phenotype, we conclude that this genetic lesion affects a gene (or genes) required for the maintenance of compaction, and propose that it be named morula decompaction (mdn). The phenotype of mdn/mdn embryos appears to be distinct from those caused by other previously described preimplantation lethal mutations or chromosomal deletions.

Animals↗
PubMed1985

X-ray microanalytical studies on cryofixed blood cells of the ascidian Phallusia mammillata. I. Elemental composition of morula cells.

Cryofixed blood morula cells of Phallusia mammillata (Cuvier), which are considered to be vanadium-accumulating cells, were examined by X-ray microanalysis using STEM (scanning transmission electron microscopy) and SEM (scanning electron microscopy). It is thought that cryopreparation preserves the native distribution of diffusible elements such as sodium, chlorine, and potassium, and prevents the displacement of vanadium, all of which may occur during conventional preparation. The results show that morula cell globules contain a large amount of sulphur and chlorine, and some sodium, magnesium, bromium and potassium, but very little or no vanadium.

Animals↗
PubMed1985

Effect of rapid addition and dilution of dimethyl sulfoxide on the viability of frozen-thawed rabbit morulae.

The aim of the present study was to examine effects of altering thawing conditions and procedure of addition and dilution of Me2SO on the viability of frozen-thawed rabbit morulae. Five hundred and sixty two rabbit morulae were cooled from room temperature to -80 degrees C at 1 degree C/min in the presence of 1.5 M dimethyl sulfoxide (Me2SO) using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, cooled rapidly, and stored in liquid nitrogen. When Me2SO was added in a single step, the frozen embryos were thawed in ambient air at 40 degrees C/min and Me2SO was diluted in a single step, 99 of 107 (93%) embryos cultured for 48 hr and 12 of 32 (38%) embryos transferred to 6 recipients developed to expanding blastocysts and viable fetuses, respectively. When Me2SO was added in a single step and the frozen embryos were thawed at the same rate and transferred directly without removal of Me2SO to culture media or oviducts of 8 recipients, 67 of 75 (89%) embryos cultured and 12 of 40 (30%) embryos transferred developed to expanding blastocysts and viable fetuses, respectively. There were no significant differences between these survival rates and survival rates obtained by conventional method, i.e., frozen embryos were thawed at 4 degrees C/min by interrupted slow method and Me2SO was added and diluted in a stepwise manner.

Animals↗
PubMed1984

Spatial distribution of blastomeres is dependent on cell division order and interactions in mouse morulae.

Spatial distribution of blastomeres was examined in 16- to 30-cell morulae obtained from aggregates of 1/2----1/2 and 1/2----2/4 blastomeres. The advanced blastomeres (2/4) contributed disproportionately more inner cells while there was a corresponding decline in the contribution from the delayed blastomere (1/2) so that a balance between the total number of inner and outer cells was retained. There was, however, no marked change in the relative number of outer cells. It is suggested that once formed, the inner more adhesive cells divide relatively faster than the outer cells whose behaviour is dictated by the inner cells. The outer less adhesive cells spread over the inner cells; cell spreading is incompatible with division. The degree to which cell spreading and retardation of division of outer cells occurs may be dictated by the number of inner cells present at any one time and this partly determines the entry of further cells inside. The suggested mechanism for cell allocation is highly flexible and, indeed, essential to encompass the wide variety of patterns of cell interactions and distribution observed in morulae. It is also proposed that the retardation of division of outer cells may trigger differentiation of trophectoderm by inducing endoreduplication and the blastomeres delayed from dividing for the longest period of time may mark down the abembryonic pole and establish the embryonic-abembryonic axis.

Animals↗
PubMed1981

The foundation of two distinct cell lineages within the mouse morula.

The division of single cells, isolated from an 8-cell mouse embryo, to give 2 x 1/16 cells has been studied by sampling cells for analysis at defined stages during and after the division. Cells were analyzed for evidence of polarity in their surface organization as assessed by fluorescent ligand binding and distribution of microvilli. Individual 1/8 cells are polarized. At division, most (82%) divide such that both the pole of ligand binding and the pole of microvilli are distributed to only one of the two daughter cells. A couplet is thereby formed with a large polar cell and a small apolar cell. Some 1/8 cells divide through the pole, generating a couplet of two polar cells, the poles being contiguous at the midbody. Elements of the surface polarity observed in the 1/8 cells can be found at all stages throughout division. Analysis of couplets of cells derived from newly formed 16-cell morulae also reveals that most consist of a polar:apolar pair and some consist of a polar:polar couplet in which the poles are contiguous at the midbody. The results indicate that two distinct cell populations are generated at division. These cells are known to occupy different positions within the morula, the polar cells being peripheral and the apolar cells being central. Since peripheral and central cells give rise to trophectoderm and inner cell mass in the blastocyst, we therefore suggest that the foundation of the trophectoderm and inner cell mass lineages may occur by a process of differential inheritance. This conclusion supports the recently proposed polarization hypothesis, which is discussed.

Animals↗
PubMed2000

Effects of cryopreservation on glucose metabolism and survival of bovine morulae and blastocysts derived in vitro or in vivo.

Bovine morulae and blastocysts were either produced in vitro through maturation, fertilization and culture of immature oocytes recovered from slaughterhouse-derived ovaries, collected in vivo or obtained after 24 h in vitro culture of in vivo collected embryos. The morulae and blastocysts were classified into four categories of embryo quality and two stages of embryonic development. Embryos were frozen by a controlled freezing method using 10% glycerol as a cryoprotectant. The ability of individual embryos to withstand freeze/thawing was measured immediately before and after cryopreservation by changes in CO2 production from (U-14C)glucose during a 2 h incubation period in a non-invasive closed system immediately before and after cryopreservation. Post-thaw survival was assessed by development in vitro during a 48 h culture period. Survival rates and oxidative metabolism after freeze/thawing were similar in embryos of the two developmental stages. However, after freeze/thawing, the rate of CO2 production of in vitro produced embryos was reduced to one half of their pre-freeze levels and was associated with poor survival rates. In vivo collected embryos had a significantly better tolerance to freezing and higher survival rates. However, when in vivo embryos were exposed to in vitro culture conditions, the rates of CO2 production and survival were significantly reduced. Pre-freeze embryo quality affected post-thaw in vitro development and metabolic activity markedly in embryos produced in vitro or pre-exposed to in vitro culture conditions. While there was no relationship between pre-freeze levels of CO2 production and post-thaw in vitro embryo development, all embryos which developed in vitro after freezing/thawing retained at least 58% of the pre-freeze levels of CO2 production regardless of their origin. Results of the present study indicate that embryos produced in vitro or pre-exposed to in vitro culture conditions are more sensitive to cryo-injury. This sensitivity is affected by embryo quality and is similarly reflected at the biochemical level. Determination of oxidative metabolism offers a feasibility for selection of viable morulae/blastocysts after freezing/thawing.

Animals↗