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PubMed1990

Effect of equilibration period on the viability of frozen-thawed mouse morulae after rapid freezing.

Mouse morulae were frozen with 1.5-4.0 M glycerol + 0.25 M lactose solution by direct plunging into liquid nitrogen vapor 0.5-30 min after equilibration at room temperature. After thawing, embryos were cultured in vitro, and the highest survival rates were obtained after exposure for 3 min at 3.0 and 4.0 M and for 5 min at 1.5 and 2.0 M glycerol levels. Significant reductions in the survival rates (P less than 0.05) were observed when equilibration periods were extended for 3-5 min at 3.0 and 4.0 M and for 5-10 min at 1.5 and 2.0 M glycerol levels. These results clearly demonstrate that the equilibration time of embryos in glycerol-lactose mixture is one of the most important factors in the present rapid freezing conditions. To clarify the factors that lower embryo viability after prolonged equilibration, we performed further experiments on the effects of exposure to glycerol-lactose mixture on the developmental potential of embryos without freezing and on the volume changes of embryos during the exposure to glycerol solution with or without lactose. It was suggested that the detrimental effects of prolonged equilibration are due not only to the toxicity and osmotic injury of higher concentrations of cryoprotectant solution but also to the influx of water into embryonic cells caused by the hypotonic salt concentration of the extracellular (freezing) solution.

Animals↗
PubMed1990

Production of chimaeric bovine embryos and calves by aggregation of inner cell masses with morulae.

Bovine inner cell masses (ICMs) were isolated by immunosurgery at day 8 or 10, or by dissection at day 14, and combined with day-5.5 morulae. Aggregation was obtained between 89%, 62%, and 0% of the day-5:day-8, day-5:day-10, day-5:day-14 composites, respectively. Chromosome analysis of composites, respectively. Chromosome analysis of composites potentially carrying the 1/29 translocation as a chromosome marker and temporarily transferred to the bovine uterus for 8 days showed that chimaeric day-14 embryos can be obtained from day-5:day-8 aggregation. The definitive transfer of eight day-5:day-8 and 11 day-5:day-10 composites resulted in the birth of six and four calves, respectively; five of the six, but none of the four, were chimaeric. The five chimaeras showed mostly the ICM phenotype. The morphological differences between ICMs at different stages of development were examined by electron microscopy and related to the success of the aggregation technique. It is concluded that bovine embryonic cells can regulate for at least 3 days difference in development but not 5 days even though aggregation is still possible.

Animals↗
PubMed1994

One-minute exposure of 4-cell mouse embryos to glucose overcomes morula block in CZB medium.

One-cell mouse embryos that block at the 2-cell stage can progress to the morula stage in CZB medium, but fail to cavitate and then swell and lyse. A 1-min exposure to 27 mM glucose at the 4-cell stage (approximately 42 hr) will support a high frequency of development to the blastocyst stage (75%) in the same medium. A glucose exposure is beneficial anytime between 30 and 54 hr of culture (67-73% blastocysts). Of a group of additional sugars and glucose analogues tested for their ability to replace glucose, only galactose was equivalent in promoting embryo development to the blastocyst stage (64% blastocysts).

Animals↗
PubMed2007

Transcription profile in mouse four-cell, morula, and blastocyst: Genes implicated in compaction and blastocoel formation.

To gain insight into early embryo development, we utilized microarray technology to compare gene expression profiles in four-cell (4C), morula (MO), and blastocyst (BL) stage embryos. Differences in spot intensities were normalized, and grouped by using Avadis Prophetic software platform (version 3.3, Strand Genomics Ltd.) and categories were based on the PANTHER and gene ontology (GO) classification system. This technique identified 622 of 7,927 genes as being more highly expressed in MO when compared to 4C (P < 0.05); similarly, we identified 654 of 9,299 genes as being more highly expressed in BL than in MO (P < 0.05). Upregulation of genes for cytoskeletal, cell adhesion, and cell junction proteins were identified in the MO as compared to the 4C stage embryos, this means they could be involved in the cell compaction necessary for the development to the MO. Genes thought to be involved in ion channels, membrane traffic, transfer/carrier proteins, and lipid metabolism were also identified as being expressed at a higher level in the BL stage embryos than in the MO. Real-time RT-PCR was performed to confirm differential expression of selected genes. The identification of the genes being expressed in here will provide insight into the complex gene regulatory networks effecting compaction and blastocoel formation.

Animals↗
PubMed1987

Effect of rapid addition and dilution of dimethyl sulfoxide and 37 degrees C equilibration on viability of rabbit morulae thawed rapidly.

The aim of the present study was to examine the effects of various conditions of addition and dilution of dimethyl sulfoxide (Me2SO) and 37 degrees C equilibration, and also the effects of freezing in the solution which was prepared in advance and stored in plastic straws at -20 degrees C on the viability of rabbit morulae thawed rapidly. The embryos were cooled from room temperature to -30 degrees C at 1 degree C/min in the presence of 1.5 M Me2SO using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, then cooled rapidly, and stored in liquid nitrogen. The frozen straws were thawed rapidly (greater than 1000 degrees C/min). When Me2SO was added in a single step, equilibrated with embryos at 37 degrees C for 15 min and diluted out in a single step, a very high survival was obtained: transferable/recovered, 90%: developed/recovered, 96%. When embryos were pipetted into 1.5 M Me2SO that was prepared in advance, stocked in straws at -20 degrees C, and cooled, the proportions of transferable and developed embryos were equivalent to those of embryos frozen in the solution that was prepared immediately before use.

Animals↗
PubMed1984

Oligosaccharides containing fucose linked alpha(1-3) and alpha(1-4) to N-acetylglucosamine cause decompaction of mouse morulae.

Two- to four-cell and eight-cell mouse embryos were incubated in various fucosylated and unfucosylated oligosaccharides, fucose binding protein, and fucosylated BSA. Compaction at the eight-cell stage was reversed by a mixture containing the oligosaccharides lacto-N-fucopentaose II (80-90%), in which fucose is linked alpha(1-4) to N-acetylglucosamine, and lacto-N-fucopentaose III (10-20%), in which fucose is linked alpha(1-3) to N-acetylglucosamine. Pure lacto-N-fucopentaose III (LNFP III) and 3-fucosyl lactose (containing fucose alpha(1-3)glucose) had a similar effect. All three molecules affected blastocyst formation. Various closely related fucosylated and unfucosylated oligosaccharides did not induce decompaction or inhibit blastocyst formation. The proportion of embryos incubated from the two- to four-cell stage in LNFP II/III which reached the eight-cell stage and formed blastocysts was reduced. Those which formed compact morulae subsequently decompacted. Precompact or early compacting eight-cell embryos incubated in LNFP II/III compacted normally but subsequently decompacted and failed to form blastocysts. Decompaction of eight-cell embryos in LNFP II/III occurred during a specific period of development (80-90 hr post-hCG) and was reversible up to 84-86 hr post-hCG, but not by 92 hr post-hCG. The period of sensitivity to LNFP II/III was associated with the decrease in the ability of calcium-free medium to cause decompaction. It appears that LNFP II/III interferes with a later calcium-independent phase of compaction and we propose that LNFP III and II inhibit an endogenous lectin-saccharide interaction between membranes involved in the stabilization of compaction.

Acetylglucosamine↗
PubMed1990

Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage.

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.

Animals↗
PubMed1985

Development and sister chromatid exchange of mouse morulae and blastocysts cultured in rat serum containing active metabolites of cyclophosphamide.

To establish an in vitro test system in which sera from animals treated with various chemicals can be tested for embryotoxic effects during the preimplantation period, mouse morulae and blastocysts were cultured in the presence of rat serum (RS) from animals which had been treated with cyclophosphamide (CPA). Development during in vitro culture for 96 h, cell number, chromosomal aberrations and sister chromatid exchange (SCE) were the end-points tested in exposed embryos. SCE frequency was the most sensitive parameter, indicating embryotoxic effects in preimplantation mouse embryos after only 1 h of exposure to RS-CPA.

Animals↗
PubMed2004

Transfer of blastocysts and morulae on day 5.

OBJECTIVE: To limit the number of embryos transferred and reduce high-order multiple pregnancies without compromising a patient's opportunity to become pregnant. DESIGN: Retrospective, nonrandomized analysis of embryo development and patient outcome when embryos were transferred on day 5. SETTING: Private practice. PATIENT(S): Women undergoing in vitro fertilization (IVF) treatment. INTERVENTION(S): Extend embryo culture to day 5 before embryo transfer (ET) to reduce the number of embryos transferred and minimize high-order multiple pregnancies. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (CPR), implantation rate (IR), and live born rate (LBR) from expanding (expanded blastocysts and blastocysts) and nonexpanding (early blastocysts and morulae) embryos transferred on day 5. RESULT(S): Approximately 60% of patients had expanding embryos (EXE) on day 5. Forty percent of patients having an ET had transferable non-expanding embryos (NEE). Patients with EXE had higher CPR and LBR compared to patients with NEE. Implantation rate and multiple pregnancy rate (MPR) were also higher for patients with EXE. The miscarriage rate (MCR) for patients with EXE and NEE was not different. Approximately 5.5% of patients did not have an ET, with most (>98%) of the ET failures from patients with <==3 two pronuclei (2PN) embryos. The number of 2PN embryos had an effect on CPR, LBR, MPR, and the number of patients having cryopreservation. CONCLUSION(S): Day 5 ET allows for the selection of embryos with the highest implantation potential as evidenced by acceptable pregnancy rates for patients with either EXE or NEE, without the need to transfer more than two embryos.

Blastocyst↗
PubMed2004

Developmental capacity of different morphological types of day 5 human morulae and blastocysts.

After prolonged culture of human embryos, the expanded blastocysts with oval inner cell mass (ICM) and cohesive trophectoderm (TE) are preferably selected for transfer. In cycles with poor embryonic development, the selection has to be done from among suboptimal blastocysts for which no grading system exists. In this study, 1396 transferred blastocysts and morulae were classified into eight morphologic categories. The B1 category constituted the optimal blastocysts. The other categories were characterized by different deviations from optimal blastocysts: cytoplasmic fragments and necrosis in TE (B2), unexpanded blastocoele (B3), non-compact or small ICM (B4), fragments in TE and ICM (B5), up to 20% excluded blastomeres (B6), necrotic TE and ICM (B7), and more than 20% excluded cells from blastocysts (B8). The live birth rate was calculated from blastocysts with known outcome after transfer (88.9% transferred blastocysts). The birth rate declined from B1 to B8 by the same order and was: 45.2, 32.8, 26.9, 23, 17.7, 16.7, 7.7 and 1.2% respectively. Normal ICM was recognized as the most important parameter for implantation. There was a strong relation between such ordered morphology categories and implantation capacity (P < 0.0001). Such a grading system is helpful in selecting the best of all available day-5 embryos for transfer.

Adult↗
PubMedDate not supplied

In vivo survival rate of rabbit morulae after vitrification in a medium without serum protein.

The in vivo survival rate of rabbit morulae after vitrification in a mixture of dimethyl sulphoxide and ethylene glycol solution without protein supplement (WPS) was compared with two types of protein supplements: rabbit serum (RS) and bovine serum albumin (BSA). Significant differences were observed in the percentage of transferable embryos (undamaged embryos after devitrification, 80.4% versus 93.2 and 92.1%, WPS, BSA and RS, respectively, P < 0.05) and live born rate (40.9% versus 56.1%, WPS and BSA, respectively, P < 0.05). Non-significant differences were, however, observed in the percentages of implanted embryos at 12 days post-ovulation induction (56.7, 69.7 and 68.6%), post-implantation survival rate (82.3, 74.2 and 77.2%) and live born rate in pregnant does (54.6, 56.1 and 50.5%) with different vitrification media (RS, BSA and WPS). We conclude that rabbit embryos can be vitrified and stored using protein-free vitrification medium with moderate losses of viability.

Animals↗
PubMed2006

Retinoids during the in vitro transition from bovine morula to blastocyst.

BACKGROUND: The conversion of retinol (ROH) to retinoic acid (RA) is crucial during development but has been not studied during blastocyst formation. METHODS AND RESULTS: In vitro-produced bovine morulae were treated for 24 h with citral (which inhibits the synthesis of RA from ROH), citral + all trans retinoic acid (ATRA), ATRA or no additives. Citral interfered with blastocyst development, whereas exogenous RA had no effect. RA, however, reversed the effect of citral on development and stimulated cell proliferation. Neither citral nor RA changed the apoptotic index, but RA triggered an increase in the apoptotic frequency of the inner cell mass. Citral and RA reduced the necrotic index. Na/K-ATPase alpha1-subunit mRNA concentrations (analysed by real-time PCR) increased after hatching and showed dependence on retinoid activity, but no evidence was found of any retinoid effect on p53 expression. Nevertheless, the p53 mRNA concentration increased in response to proliferation in hatched blastocysts. CONCLUSION: The preimplantation bovine embryo metabolizes endogenous ROH to RA, which participates in important cell processes. The true extent of the influence of RA is unknown, although the modulation of retinoid metabolism seems to be a means of increasing cell proliferation. This knowledge might be used to improve embryo quality and the efficiency of stem cell derivation.

Acyclic Monoterpenes↗
PubMed1992

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.

Animals↗
PubMed1992

Nuclear totipotency of cultured rabbit morulae to support full-term development following nuclear transfer.

The rabbit was used as a model for nuclear transfer. A critical step in nuclear transfer is oocyte activation, which was evaluated in this research. Optimal field strength of an electric stimulus for activation was examined. A significantly higher activation rate in all criteria tested was achieved when oocytes were activated electrically with a field strength of 2.4 kV/cm versus 1.2 or 1.8 kV/cm. Also, electrical stimulation with combined alternating current (AC) and direct current (DC) was superior to DC stimulation alone for activation. In another study involving 586 oocytes, exposure of oocytes to cytochalasin B for 1 h followed by activation with electrical stimulation significantly improved development of the oocytes to blastocyst stage compared to oocytes without cytochalasin B pre-exposure (38% vs 26%, p less than 0.05). Cytochalasin B exposure alone (control), however, had no effect on activation. Exposing oocytes to activation medium without electrical stimulation also activated some oocytes. In the nuclear transfer experiment, blastomeres from 8-cell embryos cultured for 20-24 h to the 32-64-cell stage were used as nuclear donor cells. Of 491 oocytes used, 459 (93%) survived the enucleation and fusion procedure, 370 (81%) fused, and 284 (77%) developed into 2-4-cell embryos. A total of 243 of these 2-4-cell embryos were transferred to 15 pseudopregnant recipients and produced 8 young (3%). Although the efficiency is low, this study demonstrated that rabbit morulae cultured for 20-24 h to the 32-64-cell stage as nuclear donors for transfer remain totipotent.

Animals↗
PubMed1999

Cryopreservation of bovine pre-morula-stage in vitro matured/in vitro fertilized embryos after delipidation and before use in nucleus transfer.

We have determined that the tolerance of in vitro matured/in vitro fertilized (IVM/IVF) bovine embryos to cryopreservation at the pre-morula stage can be improved by removal of cytoplasmic lipid droplets by centrifugation. Nucleus transfer was also performed using cryopreserved, delipated (lipid droplets removed) 8- to 16-cell-stage blastomeres of IVM/IVF embryos as donor nuclei. In vitro developmental ability of the delipated embryos to the blastocyst stage (20 of 126) was found to be equal to that of undelipated embryos (35 of 176); and of 53 delipated embryos cryopreserved at the 8- to 16-cell stage, 12 developed into blastocysts in vitro after thawing. On the other hand, only 2 of 43 undelipated embryos and 5 of 59 sham-operated embryos survived (p < 0.05). When blastomeres isolated from cryopreserved, delipated 8- to 16-cell-stage embryos were used for nucleus transfer, 57 of 80 successfully fused with enucleated oocytes, which was significantly lower than the fusion rate obtained with blastomeres of unfrozen, undelipated embryos (93 of 104, p < 0.01). However, the developmental rate to the blastocyst stage for nucleus transfer embryos reconstituted with frozen, delipated blastomeres (9 of 57) was not different from that of the nucleus transfer embryos with unfrozen, undelipated embryos (23 of 93). These results confirm that removal of cytoplasmic lipid droplets from bovine IVM/IVF zygotes allows for successful cryopreservation at the 8- to 16-cell stage and that blastomeres from these embryos can be used as donors of karyoplasts for nucleus transfer.

Animals↗
PubMed1984

Effect of parenteral administration of oestrogen and progesterone on the glycogen metabolism of mouse morulae--early blastocysts in vivo.

Morulae--early blastocysts were pulse-labelled with radioactive glucose and subsequently transferred for 24 h to ovariectomized recipients maintained on different hormone regimens. Embryos transferred to recipients treated with progesterone utilized more glycogen than did those incubated in untreated controls. Treatment with oestrogen alone had no significant effect on glycogen turnover of transferred embryos and, when given in combination with progesterone, antagonized the effect of progesterone. Priming doses of oestrogen given greater than 4 days before progesterone had no significant effect on the response to progesterone. The results indicate that the high levels of progesterone circulating in the mother before implantation cause glycogen degradation in the embryo and account for the low glycogen content of uterine embryos compared to those cultured in vitro in the presence of glucose.

Animals↗
PubMed1987

Effects of oestradiol and progesterone on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro.

The addition of progesterone (10(-7) to 10(-5) M) and/or oestradiol (10(-10) M) during 24-h chase culture of pulse-labelled morulae-early blastocysts did not affect the degradation of radiolabelled glycogen or other biochemical fractions. The presence of a high concentration of progesterone (10(-5) M) during 5-h pulse culture significantly inhibited incorporation of substrate carbon from [U-14C]glucose into both the acid-soluble and acid-insoluble glycogen fractions, but had no effect on non-glycogen fractions. Catabolic utilization of glucose as estimated by the rate of carbon dioxide and lactate production was not affected by the presence of progesterone (10(-7) to 10(-5) M), oestradiol (10(-10) to 10(-8) M) or a combination of both. The results indicate that ovarian steroids at expected physiological concentrations do not directly influence embryonic energy metabolism.

Animals↗
PubMed1990

Conversion of pregnenolone to progesterone by mouse morulae and blastocysts.

When mouse morulae, early blastocysts and implanting blastocysts were cultured with tritiated pregnenolone, tritiated progesterone and its metabolites, 5 alpha-pregnan-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one, were isolated from the medium. It appears that mouse embryos can make progesterone from pregnenolone and the progesterone is quickly metabolized into various metabolites. These abilities increase with development. It is suggested that the mouse embryo can make progesterone and may regulate its own progesterone level for optimal development.

5-alpha-Dihydroprogesterone↗