Human oviductal cells produce high molecular weight factor(s) that improves the development of mouse embryo

The coculture effects of human oviductal cells on mouse embryodevelopment in vitro were studied. Pronuclear stage mouse zygoteswere cocultured with different cell types, or then culturedeither in medium alone (control), spent medium from oviductalcell culture (conditioned medium) or high molecular weight fractions(>10 and >100 kDa) of the conditioned medium (reconstitutedmedium). Embryotrophic activities were compared between thesegroups in terms of percentage of morula and blastocyst formation,and cell count at the blastocyst stage. The mouse embryos developedbetter in oviductal cell coculture than in fibroblast cocultureand medium alone culture. Conditioned medium and its reconstitutedmedium also provided a significant enhancement of embryo developmentin vitro when compared with the control medium culture, suggestingthe production of high molecular weight embryotrophic factor(s)by the oviductal cells. The high molecular weight embryotrophicactivity accumulated with the duration of conditioning couldbe serially diluted, and was abolished by heat or trypsin treatment.Replacing bovine serum albumin with polyvinyl alcohol in theculture medium did not affect the production of this high molecularweight embryotrophic activity by oviductal cells.     

Partial purification of embryotrophic factors from human oviductal cells

Embryotrophic factors from human oviductal cells were partially purified by liquid chromatographic methods. The conditioned medium from human oviductal cell culture was fractionated successively by concanavalin A (Con-A) affinity chromatography, ion-exchange chromatography and gel filtration. The presence of the embryotrophic activity in the eluates was determined by the stimulatory effects on the development of mouse embryos in vitro. The fraction that did not bind to the lectin Con-A possessed no embryotrophic activity. Ion- exchange chromatography separated the glycoproteins that bound to Con-A into five fractions. Three of them significantly enhanced blastulation as well as conceptus formation. Gel filtration further separated these embryotrophic fractions into five fractions. Three of them with molecular weights of 154 +/- 1, 164 +/- 0.2 and 207 +/- 0.3 kDa significantly stimulated blastulation of mouse embryos. The results of this study demonstrated that several embryotrophic factors with different biochemical properties contributed to the embryotrophic effect of the human oviductal cell/mouse embryo co-culture system.      

Fertilization and early embryology: Differential effect of epithelial cell-conditioned medium fractions on preimplantation mouse embryo development

Coculture studies using preimplantation embryos have led toa number of conflicting studies. In the human, ethical considerationshave led to the preferential use of epithelial cell lines asdistinct from human Fallopian tube cells. In an attempt to isolatefactors influencing embryo development we have cultured 2-cellOF1 mouse embryos in media [Ménézo's B2 and Whittingham'sT6 supplemented with vitamins and amino acids (T6VA)] conditionedon two types of kidney epithelial cells (MBDK and Vero). Differentmolecular weight fractions of conditioned medium were used toshow the absence or presence of specific embryotrophic factors.With MDBK cells, B2 conditioned medium enhanced embryo developmentup to the blastocyst stage, while no blastocysts developed inB2 alone. When using T6VA medium, both the control and conditionedmedia showed a high percentage of blastocyst formation (57.0and 54.0% respectively), while the different molecular weightfractions showed no added improvement. With Vero cells, B2 alone,B2 conditioned medium and fractions were all detrimental toembryo development. A high percentage of blastocyst formation(between 64.7 and 75.8%) was observed in T6VA alone, T6VA conditionedmedium and fractions. Low blastocyst formation in a controlmedium can show strong positive results when medium is conditionedby cells. In contrast, a good base medium, such as T6VA, canequal the results using conditioned medium. Different cellsin contact with different types of medium show variability inthe pattern of responses, highlighting the presence of falsepositives in coculture studies.     

Insulin-like growth factor-binding proteins produced by Vero cells, human oviductal cells and human endometrial cells, and the role of insulin-like growth factor-binding protein-3 in mouse embryo co-culture systems

Co-culturing embryos on helper cells can mimic the in-vivo environment,thereby enhancing embryo development in vitro. Insulin-likegrowth factors (IGF) and their binding proteins (IGFBP) alsoenhance embryo development To investigate the kinds of IGFBPproduced by various cell monolayers and the effects of IGFBP-3on mouse embryo co-culture systems, 2-cell ICR mouse embryoswere cultured in either human tubal fluid medium alone or inthe presence of Vero cells, human oviductal cells or endometrialcells. The helper cells were analysed immunohisto-chemicallyto investigate the types of IGFBP produced by various cell monolayers.The concentrations of IGF-I and IGFBP-3 in media obtained fromthe culture of embryos alone, cells alone or cells plus embryoswere determined by radioimmunoassays. On day 7, more blastocystshatched in the co-culture groups (73% in the Vero cell group,76% in the endometrial cell group and 74% in the oviductal cellgroup) than in the control group (43%) (P < 0.0001). Theresults of immunohistochemistry revealed that (i) all threecell groups produced a lot of IGFBP-1, -2 and -3, but only alittle of IGFBP-4 and -5; and (ii) IGFBP-1, -2 and -3 were presentin blastocysts in either the presence or absence of helper cells.The IGF-I secreted by cell monolayers or embryos was undetectable(detection limit 0.83 ug/1). The IGFBP-3 concentrations in mediaobtained from co-cultured embryos and cells were significantlyhigher than in media without embryos (median values in oviductalcell culture medium, 165 versus 127 µg/1, P = 0.04; medianvalues in endometrial cell culture medium, 277.5 versus 183.5µg/1, P = 0.0002; median values in Vero cell culture medium,219 versus 120 µg/1, P = 0.011). Although IGFBP-3 concentrationin the medium that contained embryos alone was undetectableby radioimmuno-assay (detection limit 1.1 µg/1), immunohistochemistrydemonstrated the presence of IGFBP-3 in the embryos. Co-culturein systems in which there was an increased production of IGFBP-3led to an improved development of mouse embryos. IGFBP can improvethe binding of IGF to cell surface receptors of target tissue,and thus enhance the effect of limited IGF concentrations inpromoting embryo development in a co-culture system. We concludethat Vero cells, human endometrial cells and oviductal cellsproduce IGFBP-1, -2, -3, -4 and -5. IGFBP-3 may play a rolein embryotrophic potential by either regulating the action ofIGF or directly enhancing embryo development     

Embryotrophic factor-3 from human oviductal cells enhances proliferation, suppresses apoptosis and stimulates the expression of the beta1 subunit of sodium-potassium ATPase in mouse embryos

BACKGROUND: Embrytrophic factor-3 (ETF-3) from human oviductal cells enhanced the development of mouse preimplantation embryos. This report studied the embryotrophic mechanisms of the molecule. METHODS AND RESULTS: Mouse embryos were incubated with ETF-3 for 24 h at different stages of development. ETF-3 treatment between 96 and 120 h post-HCG increased the cell count of blastocysts, whilst treatment between 72 and 96 h post-HCG enhanced the expansion and hatching of the blastocysts. ETF-3 increased the cell number of the embryos by suppressing apoptosis and increasing proliferation as determined by TUNEL and bromodeoxyuridine uptake assays, respectively. Real-time quantitative PCR showed that the in vivo developed and ETF-3-treated blastocysts had a significantly higher mRNA copy number of Na/K-ATPase-beta1, but not of hepsin, than that of blastocysts cultured in medium alone. The former gene was associated with cavitation of blastocysts while the latter was related to hatching of blastocyst. The beneficial effect of ETF-3 on blastocyst hatching was also seen when ETF-3-supplemented commercially available sequential culture medium for human embryo culture was used to culture mouse embryos. CONCLUSIONS: ETF-3 improves embryo development by enhancing proliferation, suppressing apoptosis and stimulating expression of genes related to blastocyst cavitation. Supplementating human embryo culture medium with ETF-3 may improve the success rate in clinical assisted reproduction.     

Fertilization and early embryology: Established cell lines and their conditioned media support bovine embryo development during in-vitro culture

These experiments were conducted to evaluate the ability ofdifferent somatic–cell monolayers or conditioned mediumfrom somatic cells for supporting bovine embryo developmentin vitro. In the first experiment, bovine embryos (2- to 4-cells)were allocated randomly to a control (medium 199 with 10% fetalbovine serum and antibiotics) group or co-cultured with bovineoviduct epithelial (BOEC), buffalo rat liver (BRL), Madin Darbybovine kidney (MDBK) or African green monkey kidney (Vero) cells.In the second experiment, bovine embryos (1-cell) were allocatedrandomly to the following groups: control medium or conditionedmedium from BOEC, BRL, MDBK and Vero monolayers. In both experiments,development to the blastocyst stage was assessed after 8 daysof incubation at 39°C and 5% CO₂ In Experiment 1, co-cultureimproved development to the blastocyst stage compared with controlmedium alone, and the highest development was observed afterco–culture with BOEC. In Experiment 2, conditioned mediumenhanced development to morulae and blastocysts compared withthe control medium; however, no differences were detected amongdifferent cell supports. These results indicate that both co-cultureand conditioned medium from different cell monolayers supporteddevelopment to the blastocyst stage at a higher efficiency thancontrol medium alone.     

Fertilization and early embryolgoy: Can Matrigel substitute for Vero cells in promoting the in-vitro development of mouse embryos?

The influences of Vero cells and the basement membrane substratumfor these cells (Matrigel^®) on the rate of hatched blastocystformation from mouse zygotes in vitro were compared. Zygotesobtained from C57BL/6xBALB/c F1 females pretreated with pregnantmare's serum gonadotrophin/human chorionic gonadotrophin matedwith BDF1 males were cultured (120 h) in human tubal fluid mediumsupplemented 0.5% with bovine serum albumin. The rates of earlyhatching and hatched blastocyst formation at 96 and 120 h ofculture were expressed as the percentage of 2-cell embryos visualizedafter the initial 24 h. The rate of total blastocyst formationdid not differ between treatment groups. However, <10% ofembryos cultured for 96 h in medium alone advanced to the hatchingstage compared with 35–40% of blastocysts cultured withVero cells or with Matrigel alone. Similarly, by 120 h of culture,only 20% of embryos cultured in medium alone developed to hatchingor hatched blastocysts compared with >70% for those embryosco-cultured with Vero cells or with Matrigel. In conclusion,Vero cells improved the rate of development of mouse embryosto hatched blastocysts during serum-free culture. Similar improvementswere seen in the presence of Matrigel alone; Matrigel is thebasement membrane substratum used for the Vero cells. Furtherstudies on the means whereby Matrigel promotes early embryonicdevelopment (e.g. appropriate combination of basement membrane-associatedgrowth factors) may lead to a safe, defined medium preparationfor the stimulation of in-vitro development of human embryos.     

Fertilization and early embryology: Nuclei number in human embryos co-cultured with human ampullary cells

A study was undertaken to evaluate the effect on nuclei numberin human embryos cultured in vitro with primary cell lines ofhuman Fallopian tube epithelium. The development of 203 surplushuman embryos, cultured in standard culture medium (Earle'sbalanced salt solution+15% A5) with or without ampullary cells,was observed from day 2 to day 5.5 post-insemination. The expandedblastocysts in both culture conditions were analysed for nucleinumbers per blastocyst. Embryos transferred to co-culture atthe 2-cell stage had an average of 120.7 nuclei per blastocyst,which was significantly higher than the average of 62.9 nucleiper blastocyst (P=0.023) for the embryos transferred to co-cultureat the 4-cell stage. The embryos cultured in the control mediumhad an average of 42.1 nuclei per blastocyst, which was significantlyless than co cultured embryos (P=0.04). Severely fragmentedembryos (grades 3 and 4) did not show recovery in co-culture.Our results show that when human embryos are transferred toco-cultures before the 4-cell stage, the blastulation rate andthe cell number per embryo increase significantly compared tothe embryos cultured in standard culture medium. The possibleeffect of co-culture on embryonic gene expression is discussed.     

Use of Synthetic Serum Substitute and alpha-minimum essential medium for the extended culture of human embryos to the blastocyst stage

This study was designed to provide further information on mouse and human embryo development in alpha-modified minimum essential medium (alphaMEM). First, we compared the development and implantation potential of murine in-vitro fertilized (IVF) embryos cultured in alphaMEM, in the presence and absence of co-culture cells. No significant difference was observed in blastocyst rate between alphaMEM alone (76.2%) and alphaMEM plus co-culture (79.9%). The percentage of hatched blastocysts was, however, higher with co-culture (47.5 versus 40%, P < 0.01). Transfer of blastocysts to pseudopregnant foster mothers resulted in similar live birth rates (14.9% alphaMEM alone versus 19.8% alphaMEM/co-culture). alphaMEM was also introduced into our clinical IVF programme for culture of human embryos beyond day 3. Spare human embryos were cultured under oil in microdrops of alphaMEM supplemented with 10% synthetic serum substitute. Blastocysts were evaluated for maturity and the presence and organization of the inner cell mass. A total of 206 embryos from 53 IVF patients underwent extended culture. The overall blastocyst rate was 45.1%. An inner cell mass was observed in 76 blastocysts (81.7%). With regard to developmental maturity, approximately 73% of blastocysts that had been frozen were expanding (cavity > 50% embryo volume) or fully expanded. These data suggest that alphaMEM in conjunction with a commercial protein preparation such as Synthetic Serum Substitute may be a good basal medium for culture of human embryos to the blastocyst stage.      

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.     

Preliminary clinical experience with human blastocyst development in vitro without co-culture

This preliminary analysis was designed to quantify blastocyst development of supernumerary embryos without the use of feeder cells, conditioned medium or whole serum. Embryos derived from in-vitro fertilization (IVF) that were not transferred or cryopreserved were included in this study. Ova were harvested for IVF after a standard ovarian stimulation with gonadotrophin-releasing hormone agonist/ human menopausal gonadotrophin (GnRHa/HMG) or follicle-stimulating hormone (FSH). Ova were collected and culture in 150 microliters droplets of P1 medium under mineral oil, in groups at 37 degrees C under 5% CO2, 5% O2, 90% N2 (group A) or under 5% CO2 in air (group B) environment. Embryo transfer was performed 72 h post-harvest. Viable embryos not transferred or cryopreserved were placed in blastocyst medium and cultured for an additional 48 h in 5% CO2 in air. Embryos that exhibited an expanded blastocoelic cavity and well-defined inner cell mass at 120 h were counted. Of 838 supernumerary embryos cultured, 448 (53.5%) reached the expanded blastocyst stage by 120 h of culture. Patients were given the option of cryopreservation at that time. The embryos were cryopreserved using a standard protocol with serial addition of glycerol. Embryos reaching the blastocyst stage after more than 120 h of culture were not included. There was no difference in the proportions of blastocyst development between group A, 217/410 (53.5%) and group B, 231/428 (54%). To date, 16 patients have each had up to three thawed blastocysts transferred, out of whom seven became pregnant. This report demonstrates that a simple system of sequential culture generated acceptable, viable blastocyst development (54%) with supernumerary embryos, without the use of feeder cells, conditioned medium or whole serum. Recognizing the differential metabolic requirements of early and late cleavage stage embryos has enabled the application of a glucose/phosphate-free simple culture medium (P1) for up to 72 h of culture and a complex, glucose-containing medium (blastocyst medium) for subsequent blastocyst development.     

Improved development of human embryos in vitro by a human oviductal cell co-culture system.

This study was undertaken to determine the effect of co-culture with human oviductal cells on human embryos. Spare embryos from gamete intra-Fallopian transfer (GIFT), pronuclear stage transfer (PROST) and in-vitro fertilization/embryo transfer (IVF/ET) programmes were either cultured in serum-supplemented Earle's balanced salt solution alone, or co-cultured in the same solution with oviductal cells from the pronuclear stage (day 1 post-insemination) or two- to four-cell stage (day 2 post-insemination). The co-cultured embryos appeared to have a higher developmental potential (higher rate of blastocyst formation and lower fragmentation rate), although there was no statistical difference in their rate of development, degree of fragmentation and stages attained, when compared with conventionally cultured embryos. The percentage of hatching blastocysts was significantly higher (P less than 0.05, Fisher's exact test) for embryos co-cultured from day 1 post-insemination (38%) than for embryos which had not been co-cultured (7%). The blastocyst hatching rate for embryos co-cultured from day 2 post-insemination was 15%. It was therefore concluded that co-culture of human embryos with oviductal cells could improve the development of the embryos in vitro. The degree of improvement was more pronounced when the co-culture started at an earlier stage.     

Fertilization and early embrology: Vero cell effect on in-vitro human blastocyst development: preliminary results

Asynchrony between embryo and uterine environment is one ofthe major limits in human in-vitro fertilization (TVF). A culturesystem which could prolong culture time and increase embryoniccleavage rate and viability would improve success rates. UsingVero cells, an in-vitro co-culture system was developed to investigateand promote human embryo development. Vero cells provide goodsupport for human early embryos up to the blastocyst stage.When fertilized embryos were co-cultured, 68% of them reachedthe blastocyst stage. Pregnancy rate was 50% per transfer inpatients with several previous failures of implantation. A significantincrease in clinical pregnancy rate was also demonstrated whenzygotes were maintained on Vero cell monolayer for only 24 h.The beneficial effect of the feeder layer may be through therelease of embryotrophic factors and the detoxification of theculture medium by the cells. Co-culture is a new concept inassisted reproduction.     

Effect of cryoprotectants and their concentration on post-thaw survival and development of rapid frozen-thawed pronudear stage mouse embryos

Experiments have been conducted to develop a simple rapid-freezingprotocol for pronudear stage mouse embryos. The effect of typeof cryoprotectant (dimethyl-sulphoxide (DMSO) or 1,2-propanediol(PROH)) and concentration (ranging from 3.5 to 8.0 mol/1 with0.5 mol/1 sucrose) on the post-thaw morphological survival rate,on cleavage rate and on development to the blastocyst stagewere studied. Further, in-vivo viability of embryos frozen usingthe most effective cryoprotectant concentration (PROH at 7.0mol/1) was compared with viability of non-frozen embryos. Thetype of cryoprotectant and its concentration influenced thesurvival and development of embryos to the blastocyst stagein vitro. The best development with PROH was achieved at 7.0mol/1 (66%, 128/193), whereas with DMSO the best developmentwas achieved at 6.0 mol/1 (42%, 71/171). The rates of survivaland cleavage did not differ between the two best cryoprotectantconcentrations (P > 0.01) but the proportion of embryos whichdeveloped to blastocyst was higher (P > 0.001) with PROHat 7.0 mol/1 compared with DMSO at 6.0 mol/1. The rates of survivaland development were higher (P < 0.001) with DMSO at 3.5and 6.0 mol/1 compared with similar concentrations of PROH.The cleavage and development, however, was higher (P < 0.001)at 7.0 mol/1 PROH compared with the same concentration of DMSO.At 8.0 mol/1 the survival and development was not different(P > 0.01) between DMSO and PROH. The rate of implantationand the percentage of live fetuses at autopsy of the recipientsreceiving non-frozen embryos was higher (63 and 41% respectively)than in those receiving frozen–thawed embryos (53 and37% respectively), but not significantly different. It may beconcluded that the concentration range of cryoprotectants whichallows acceptable embryo viability after freezing and thawingis very narrow. The rapid protocol using dehydration in 0.25mol/1 and 0.5 mol/1 sucrose followed by exposure to 7.0 mol/1PROH and 0.5 mol/1 sucrose for 45 s was the most effective forcryopreservation of pronudear stage mouse embryos.     

Fertilization and early embryology: Improved cleavage rate of human embryos cultured in antibiotic-free medium

Retarded development and blastomere fragmentation of human prelimplantationembryos represent a common phenomenon in in-vitro culture systems.Even though media composition is generally formulated to meetembryo nutritional requirements, the influence of antibioticsupplementation has not been investigated thoroughly. The presentstudy was performed to evaluate the effects of antibiotics onembryo morphology and growth in modified culture media. A totalof 196 zygotes from 18 couples was cultured in three differentmedia: (i) conventional medium (n = 99, control group); (ii)medium modified with half the standard antibiotic concentration(n = 54); and (iii) antibiotic-free medium (n = 43); 49 embryosfrom the control group were selected at the zygote stage andtransferred to the patients on day 2. The remaining 147 zygoteswere cultured to the blastocyst stage for cryopreservatlon;their morphology and cell number were assessed daily at 40,64, 88, and 112 h post-insemination. Overall cleavage rate was95% and embryo scoring revealed 91% grade 1 embryos throughoutthe culture period in the three media. Significantly highercleavage rates were obtained in the antibiotic-free medium ateach observation, including the blastocyst stage, when comparedto the other two groups. In addition, no notable improvementwas observed in the embryos cultured in a reduced concentrationof antibiotics. In conclusion, antibiotic supplementation ofmedia has an adverse effect on the growth rate of preimplantationembryos, even in reduced concentrations, suggesting that antimicrobialdrugs may interfere with the timing of cleavage events eitherby delaying or blocking embryo development.     

Evaluation of Vero cell co-culture system for mouse embryos in various media.

Our objective was to evaluate and compare the efficacy and mechanisms of co-culturing mouse embryos with Vero cells in both Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) and human tubal fluid (HTF) culture medium. Two-cell CB6F1 mouse embryos were cultured either in the presence of Vero cells (group A) or in culture medium alone (group B). In DMEM/F12 significantly more morulae developed in group A than in group B on day 3 (91 versus 23%; P less than 0.01). In contrast, the mouse embryos grew rapidly in HTF and significant differences were noted only in later embryonic stages (on day 5; 86% and 50%, P less than 0.01 of group A and B respectively, hatching or hatched). Similar experiments using DF1 and ICR mouse strains also revealed enhanced embryo development in the presence of Vero cells. To determine whether the embryo-enhancing effects of Vero cells were due to the removal of toxins or to the secretion of embryotrophic factors, ICR mouse embryos were cultured in fresh media with cells (group A), without cells (group B) and in cell-free conditions using cell-conditioned media which were obtained in the presence (group C) or absence (group D) of embryos. These results demonstrated that completion of hatching was highest (52%; P less than 0.01) in group A after 6 days in culture. There were no significant differences between groups B, C and D (rates of total hatching 18, 17 and 17%, respectively). It is concluded that Vero cells improve the development of mouse embryos and this is likely to be due to removal of substances inhibitory to development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of different biopsy methods of blastomeres from 2-cell mouse embryos

As an outgrowth of in-vitro fertilization and embryo transfer,detection of genetic and metabolic defects prior to implantation might be possible in the future. The objective for preimplantationdiagnosis would be to sample a minimum of cell material of theconceptus for diagnosis prior to transfer. Different protocolsfor isolating Individual blastomeres out of 2-cell mouse embryoswere evaluated. 2-cell mouse embryos (from F₁ hybrids C57B1females x CBA males) were collected and the zona pellucida wasremoved by enzyme treatment (pronase), by exposure to Ca²⁺ -Mg²⁺-free acid Tyrode (pH = 2.5) or by mechanical force. Individualblastomeres were obtained by exposure to an enzyme (pronase),to a chelating agent (EDTA-glycine mixture), to Ca²⁺ -Mg²⁺-freePBS or after isolation by mechanical force. The biopsied blastomereswere then cultured in vitro as such or first replaced into ahost zona pellucida. Evaluation was perfonned by culture invitro up to the blastocyst stage and by transfer of embryosappearing morphologically normal into pseudopregnant fostermothers. A chromosomal study of the second mitotic divisionof the isolated blastomere was also performed. All isolationprocedures had a negative impact on the In-vitro and in-vivogrowth patterns of the isolated blastomeres. After culture invitro to the blastocyst stage, different abnormalities couldbe observed: embryos lacking compaction, embryos with doubleblastocoelic cavities and embryos with no inner cell mass (trophoblasticvesicle). After replacement of the isolated blastomeres intoa host zona pellucida, similar observations could be made. Chromosomalanalysis did not reveal a clear influence of the different biopsymethods on the mitosis of the isolated blastomeres.     

Assessment of growth factor effects on post-thaw development of cryopreserved mouse morulae to the blastocyst stage

The objective of this study was to assess the influence of specific factors on post-thaw development of mouse cryopreserved morulae. Thawed morulae (n = 206) were randomly distributed between 10 treatment groups: medium alone control (CT), Vero (VR) cells, leukaemia inhibitory factor (1 ng/ml), interleukin-6 (1 ng/ml), transforming growth factor (TGF) alpha (2 ng/ml), epidermal growth factor (EGF) (4 ng/ml), platelet-derived growth factor (1 ng/ml), insulin-like growth factor (IGF)-I (30 ng/ml), IGF-II (1 ng/ml) and TGFbeta (2 ng/ml). At 4, 8, 20, 30 and 48 h, a digitized image of each thawed embryo was captured and stored for later analysis. The following parameters were examined: blastocoel formation, blastocyst expansion, zona thickness and hatching. At termination of the experiment, cell number per embryo was determined by bisbenzimide staining. When contrasted to the medium alone control, co-culture consistently accelerated the development of frozen-thawed morulae to the hatched blastocyst stage, allowing embryos to recover rapidly from any damage sustained during the cryopreservation process. While no single growth factor/cytokine was able to completely mimic the results achieved with co-culture, all of the growth factors impacted positively on at least one of the morphological parameters studied. Cell proliferation was significantly stimulated by just 48 h exposure to growth factors, either through co-culture or by direct media supplementation. Co-culture again yielded the best results with a mean cell count of 217 +/- 76 cells per blastocyst as compared with 131 +/- 36 in control medium alone. Amongst the factors tested, IGF-I, IGF-II and EGF had the greatest impact, with mean cell counts of 172 +/- 50, 168 +/- 50 and 179 +/- 55 respectively. Whereas only 5% of CT embryos developed to blastocysts with > 200 cells, 51% of thawed embryos placed on co-culture monolayers and 25-32% of embryos cultured with IGF-I, IGF-II or EGF had > 200 cells. This study for the first time systematically describes the effect of culture regimen and growth factor additives on the post-thaw development of cryopreserved embryos.     

小鼠1-细胞胚胎体外培养条件的研究

目的　检验培养基（Ｗｈｉｔｔｅｎ、ＣＺＢ、ＳＯＭ 和ＫＳＯＭ）、培养器皿（进口、国产新旧平皿）与配制培养基的水质对小鼠胚胎体外发育的影响。　方法　采用微滴培养法培养昆明种小白鼠１－细胞胚胎,以胚胎发育到囊胚的比例为判断培养效果的标准。　结果　Ｗｈｉｔｔｅｎ、ＣＺＢ、ＳＯＭ 和ＫＳＯＭ 培养基中２－细胞发育率分别为１００％ 、１００％ 、１００％ 和９８．６％ ,而囊胚发育率却分别为０％ 、７８．３％ 、４．８％ 和９．５％ ;用三蒸水、五蒸水和去离子水配制的ＣＺＢ培养基,培养囊胚发育率分别为４０．８％ 、５４．１％ 和５２．４％ ;国产和进口新平皿的囊胚发育率分别为６８．０％ 和７２．２％ ;国产和进口旧平皿的培养囊胚发育率为２７．２％ 和４６．１％ 。　结论　昆明小白鼠早期胚胎存在体外发育阻断现象;ＣＺＢ培养基培养昆明小白鼠早期胚胎效果最好;用三蒸水、五蒸水和去离子水配制的ＣＺＢ培养基培养结果无显著差别;进口和国产新平皿的培养效果相近,但再次使用时培养效果显著下降。