Trophectoderm growth and bilateral symmetry of the blastocyst in the mouse

BACKGROUND: The present study was undertaken to ascertain whether the polarized flow of cells from polar to mural trophectoderm is related to the axis of bilateral symmetry of the blastocyst in the mouse, and whether trophectoderm cells can initiate new cycles once they have left the polar region. METHODS AND RESULTS: Two different approaches were used to investigate the relationship of polar to mural flow of trophectoderm cells to the bilateral axis. One was to mark peripheral polar trophectoderm cells at one or both ends of the bilateral axis in early blastocysts and examine the distribution of their clonal descendants after further growth in culture. The other was to mark the two ends of the bilateral axis with small oil drops in the zona pellucida in blastocysts whose polar trophectoderm was then labelled globally with fluorescent latex microspheres before culture. In both cases, marking of additional blastocysts orthogonal to the bilateral axis was also done. The results show that the direction of polar to mural flow of cells is not random, and that the most distal mural trophectoderm cell could yield up to eight descendants during 45 h of culture. CONCLUSION: The findings are consistent with the polar to mural flow of trophectoderm cells being aligned with the bilateral axis. Moreover, trophectoderm cells can embark on new cycles even when remote from the inner cell mass.     

Possible involvement of crosstalk cell-adhesion mechanism by endometrial CD26/dipeptidyl peptidase IV and embryonal fibronectin in human blastocyst implantation

When human blastocysts hatch through the zona pellucida, gaining the ability to adhere to the endometrium, crosstalk between the embryo and the uterus may represent a successful outcome of their synchronized development and differentiation. CD26/dipeptidyl peptidase IV is known as a marker molecule of the implantation phase endometrium. To study the role of CD26 in implantation, 35 human hatched blastocysts were prepared by enzymatic treatment of expanded blastocysts that had been grown on schedule from frozen-thawed surplus embryos at the 2- or 4-cell stage. The blastocysts were placed on CD26-overexpressing or mock-transfected control monolayer cell cultures. The CD26-overexpression caused significantly higher blastocyst adhesion rate (53.3% versus 25.0%, P < 0.05) and significantly larger outgrowth area of trophectoderm (1.7-fold, P < 0.05). The second part of the present study was to show the expression of fibronectin, a CD26 ligand, in human preimplantation embryos, using the same donated resources. Fibronectin mRNA was detected by RT-PCR from the single hatched blastocyst (2/2) and from the single early blastocyst (3/6) but not from the single morula (0/5) samples. An indirect immunofluorescence technique verified the localization of fibronectin on the surface of the blastocyst. These results indicate that the adhesion mechanism by endometrial CD26 and embryonal fibronectin may be involved in human blastocyst implantation.     

Flow of cells from polar to mural trophectoderm is polarized in the mouse blastocyst

During growth of the blastocyst there is a net flow of cells from the polar to the mural trophectoderm which is presumed to be radially symmetrical. However, such a pattern of cell movement is inconsistent with findings from a recent clonal analysis. To visualize the overall flow of cells directly, the polar trophectoderm of expanding blastocysts was labelled globally with fluorescent microspheres. Following further growth, the great majority of blastocysts that remained labelled throughout the polar trophectoderm exhibited a polarized rather than radial spread of label into the mural region. This was the case regardless of the labelling technique, whether the blastocysts were grown in utero or in vitro, or had the zona pellucida removed or left on. Intriguingly, where there were two foci of spread of label into the mural trophectoderm rather than one, these were diametrically opposite each other. In further experiments, fluorescent lineage labels were used to distinguish junctional trophectoderm cells with and without an extension onto the blastocoelic surface of the inner cell mass. The location of clones formed following further blastocyst growth provided no evidence that egress of cells from the polar trophectoderm is restricted circumferentially by the presence of junctional cells having an extension.     

Basement membrane and fibronectin matrix are distinct entities in the developing mouse blastocyst

Immunocytochemical techniques were used to study the distribution of fibronectin, type IV collagen (collagen-IV), and laminin in four different stages of mouse blastocyst development. Immunoreactivity for collagen-IV and laminin is present in a granular pattern inside the inner cell mass (ICM) cells in stage 1 blastocysts, while these blastocysts are negative for fibronectin. Fibronectin immunoreactivity appears extracellularly under the trophectoderm (TE) in stage 2 blastocysts, in the form of homogeneously distributed dots, and/or fibrils located preferentially close to cell boundaries. It is followed by the appearance of both collagen-IV and laminin immunoreactivity in patches on the basal side of the TE in stage 3 blastocysts. These patches are initially localized under the central region of TE cells, thus in a location clearly different from that of fibronectinpositive fibrils. As development proceeds the collagen-IV- and laminin-positive patches become larger, covering, by stage 4, an extensive portion of the inner lining of the blastocoel. Fibronectin-positive material is still present in a fibrillar form in stage 3 blastocysts, but is generally reduced to thin strands by stage 4. These results indicate that fibronectin is independent of the mouse blastocyst basement membrane, but may play a transient role in cell adhesion during its deposition. In addition, the results suggest that the ICM plays a major role in the production of collagen-IV and laminin, while the basal surface of TE cells is the primary site of basement membrane assembly.     

Basement membrane and fibronectin matrix are distinct entities in the developing mouse blastocyst.

Immunocytochemical techniques were used to study the distribution of fibronectin, type IV collagen (collagen-IV), and laminin in four different stages of mouse blastocyst development. Immunoreactivity for collagen-IV and laminin is present in a granular pattern inside the inner cell mass (ICM) cells in stage 1 blastocysts, while these blastocysts are negative for fibronectin. Fibronectin immunoreactivity appears extracellularly under the trophectoderm (TE) in stage 2 blastocysts, in the form of homogeneously distributed dots, and/or fibrils located preferentially close to cell boundaries. It is followed by the appearance of both collagen-IV and laminin immunoreactivity in patches on the basal side of the TE in stage 3 blastocysts. These patches are initially localized under the central region of TE cells, thus in a location clearly different from that of fibronectin-positive fibrils. As development proceeds the collagen-IV- and laminin-positive patches become larger, covering, by stage 4, an extensive portion of the inner lining of the blastocoel. Fibronectin-positive material is still present in a fibrillar form in stage 3 blastocysts, but is generally reduced to thin strands by stage 4. These results indicate that fibronectin is independent of the mouse blastocyst basement membrane, but may play a transient role in cell adhesion during its deposition. In addition, the results suggest that the ICM plays a major role in the production of collagen-IV and laminin, while the basal surface of TE cells is the primary site of basement membrane assembly.     

Differentiation of ICM cells into trophectoderm

It has been established previously that when inserted in the blastocyst E Ca 247 preferentially differentiates into trophectoderm in vitro. If the concept that tumors are caricatures of the process of tissue renewal is correct, then some cells from the inner cell mass (ICM), the normal counterpart of embryonal carcinoma, should be able to differentiate into trophectoderm. This has been a controversial issue. Four experiments are now reported that support the idea that ICM can differentiate into trophectoderm: 1) ICM from early blastocysts after classical immunosurgery made blastocysts in vitro; 2) ICM obtained from early blastocysts by immunosurgery using antigens other than histocompatibility ones made blastocysts in vitro; 3) ICM from early blastocysts, in which the trophectodermal cells had been labeled, contained no labeled cells following immunosurgery; and 4) In reconstruction experiments, polar and mural trophectodermal cells attached to ICM from late blastocysts failed to multiply and make blastocysts when cultured. It is concluded that like the embryonal carcinoma some ICM cells of early blastocysts have the potential to make trophectoderm. This fact is consistent with the concept that tumors are caricatures of the process of tissue renewal; and establishes E Ca 247 as a good model for study of trophectodermal differentiation.     

Retinoic acid decreases the viability of mouse blastocysts in vitro

BACKGROUND: This study was designed to examine the cytotoxic effect of retinoic acid on the blastocyst stage of mouse embryos and on subsequent early postimplantation embryo development in vitro. METHODS AND RESULTS: Mouse blastocysts were exposed for 24 h to doses of 0, 0.1 micromol/l and 10 micromol/l all-trans retinoic acid and observed for their capacity to implant and develop during the early postimplantation period in vitro. When retinoic acid-pretreated blastocysts were allowed to implant in vitro, significantly fewer embryos were able to reach a later stage of embryo development. Compared with the findings for the control blastocysts, exposure to retinoic acid resulted in a significant reduction in the average number of total cells in blastocysts and the trophectoderm/inner cell mass lineage. The effect was associated with a significant increase in the frequency of cells identified as being engaged in apoptosis by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling and Annexin V techniques. CONCLUSIONS: This is the first evidence that retinoic acid induces cell death (apoptosis) and inhibits cell proliferation in mouse blastocysts. This results in the retardation of early postimplantation blastocyst development and subsequent blastocyst death.     

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.     

Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.     

Blastocyst development after assisted reproduction using spermatozoa obtained after testicular stem cell transplantation in mice

BACKGROUND: Since its introduction in 1994, testicular stemcell transplantation (TSCT) has been widely used for research.This technique may also become important for preserving fertilityin pre-pubertal cancer patients. Therefore, it is necessaryto investigate the safety aspects of reproduction using spermatozoaobtained after TSCT. In this study, preimplantation developmentof mouse embryos, using spermatozoa obtained after TSCT, wasexamined. METHODS: TSCT-derived spermatozoa were used for IVFand ICSI. Embryos were cultured for five days until they reachedblastocyst stage and were evaluated by differential staining.RESULTS: IVF revealed significantly lower fertilization anddevelopment rates after TSCT-IVF compared to control-IVF. Blastocystsderived from TSCT-IVF had significantly lower inner cell massnumbers (ICMs) and lower ICM/trophectoderm (TE) ratios comparedto control-IVF blastocysts. No differences in fertilizationand development rates were observed between TSCT-ICSI and control-ICSI,and blastocyst quality in the transplanted group was similarto that of the control blastocysts. CONCLUSION: Our study showedthat after TSCT-IVF, fertilization and preimplantation developmentwere disturbed and blastocysts showed reduced ICM and ICM/TEratio. However, after TSCT-ICSI, both fertilization and preimplantationdevelopment were normal and blastocyst formation was comparableto control-ICSI.     

Co-culture of 1-cell outbred mouse embryos on bovine kidney epithelial cells: effect on development, glycolytic activity, inner cell mass: trophectoderm ratios and viability

In an attempt to enhance embryo development, we have co-cultured1-cell OF1 mouse embryos on bovine kidney epithelial (Madine-Darbybovine kidney; MDBK) cells in a complex medium called complexmouse tubal fluid (cMTF; based on the energy substrate levelsfound in the mouse oviduct, containing non-essential amino acids,glutamine and EDTA). To determine the quality of the blastocystsobtained, we examined several parameters: morphology, totalcell numbers, inner cell mass (ICM): trophectoderm (TE) ratio,glycolytic activity and viability after transfer. A significantlylower number of blastocysts developed on MDBK cells comparedwith cMTF medium. cMTF blastocysts had a significantly higherglycolytic activity and a lower blastocyst cell number thanthose grown in co-culture, while both in-vitro groups had higherICM: TE ratios compared with in vivo. Blastocysts grown on MDBKcells displayed an elevated ICM number compared with those grownin cMTF medium alone. However, the percentage of fetuses aftertransfer remained drastically low in both culture groups comparedwith in-vivo blastocysts. In conclusion, co-culture did notincrease the number of zygotes reaching the blastocyst stage.Although co-culture blastocysts show some similarities to in-vivoembryos in cell number and glycolytic activity, no enhancementin viability was observed.     

Mouse embryo development following IVF in media containing either L-glutamine or glycyl-L-glutamine

BACKGROUND: The development of the mouse zygote following fertilization in vitro in a KSOM-type medium containing either L-glutamine or glycyl-L-glutamine has been examined, and compared with the development of mouse zygotes produced by natural fertilization. METHODS: Mouse IVF, embryo culture and embryo transfer. RESULTS: Fertilization rates, development to the blastocyst stage, implantation rate, gross fetal development and fetal body weight are not different in a KSOM-type medium containing either L-glutamine or glycyl-L-glutamine. No evidence of abnormal fetal development, such as exencephaly, was observed. The replacement of L-glutamine with glycyl-L-glutamine favoured the development of relatively more inner cell mass cells than trophectoderm cells, and reduced the numbers of pyknotic and fragmented nuclei in the blastocysts that developed in vitro. CONCLUSIONS: There is no evidence that the presence of glutamine in the medium used for IVF influences significantly the subsequent development of the zygote. Replacing glutamine with glycyl-L-glutamine may be advantageous.     

Fertilization and early embryology: Clonal analysis of growth of the polar trophectoderm in the mouse

The results of experiments in which horseradish peroxidase (HRP)was used to mark single trophectoderm or inner cell mass (ICM)cells in situ in mouse blastocysts have led to the proposalthat growth of the trophectoderm depends on stem cells locatedin the inner cell mass. Thus, the finding that the visual centreof clones formed following labelling of the central polar trophectodermcell in early or expanding blasrtocysts was consistently shiftedtowards or into the mural trophectoderm was attributed to theirdisplacement by ICM-derived cells. However, the frequency withwhich central polar cells were displaced is likely to have beenoverestimated by using the visual centre of descendant clonesas the index of their location. also, the possibility that displacementof central polar cells was an artefact of the marked temporaryinterruption of their cycling that resulted from labelling wasnot discounted. Furthermore, no attempt was made to ascertainwhether cells located elsewhere in the polar trophectoderm alsomoved murally, as expected if there is a general displacementof such cells. In the present study, labelling of either thecentral or a peripheral polar trophectoderm cell with HRP wasachieved without obviously perturbing their subsequent proliferation.Moreover, displacement was assessed by recording the locationof the proximal boundary rather than the visual centre of theresulting clones. Even by this conservative criterion, the majorityof labelled central cells moved towards or into the mural trophectoderm. In marked contrast, however, labelled peripheral polarcells moved murally in only a minority of cases. The remaindereither retained their original position or moved towards ratherthan away from the central polar region. Such an anisotropicpattern of growth of the polar trophec toderm is not readilyexplicable in terms of recruitment of cells from the ICM. Rather,it accords with the view that the polar trophectoderm is a proliferativecentre, and suggests that movement murally of its surplus cellsmay be restricted circuinferentially, possibly through anchorageof the junctional trophectoderm cells that extend processesover the free surface of the ICM.     

Slow controlled-rate freezing of sequentially cultured human blastocysts: an evaluation of two freezing strategies

BACKGROUND: To optimize blastocyst cryopreservation, the prerequisite is to develop a better understanding of factors that influence their survival and implantation potential. Therefore, the aim of the present work was to evaluate, retrospectively, the outcome of blastocyst cryopreservation in a day 2/3 fresh embryo transfer programme. METHODS: Two different freezing strategies were compared: a first strategy (strategy A: 3007 blastocysts frozen) consisted of freezing those blastocysts that had at least a cavity; a second strategy (strategy B: 3831 blastocysts frozen) consisted of freezing only more advanced stage blastocysts with a good quality inner cell mass and trophectoderm. The outcome of cryopreservation, as related to the two different freezing strategies, was analysed. In addition, after freezing and thawing, we evaluated the influence of blastocyst developmental characteristics on immediate morphological survival and further development in vitro. RESULTS: The immediate morphological survival after thawing was higher for early blastocysts as compared to advanced and hatching blastocysts. The further developmental potential in vitro of thawed blastocysts was higher for advanced and hatching blastocysts as compared to early blastocysts. As a result, the percentage of deliveries, calculated as a percentage of started thawing cycle, and the percentage of children born, calculated as a percentage of embryos transferred, was not different for strategies A and B. CONCLUSION: The results clearly indicate that culture conditions and cryopreservation procedures of blastocysts need to be further improved.     

Fertilization and Empryology: The effect of epidermal growth factor on growth and differentiation of mouse preimplantation embryos in vitro

The effect of epidermal growth factor (EGF) on embryonic growth,development, attachment and spreading in vitro was studied.EGF was added to 130 embryos at the 4-cell stage; to 128 embryosat the blastocyst stage; and to 147 embryos 24 h following spreading.Development of embryos from the 4-cell to the blastocyst stage,differentiation of the inner cell mass (ICM) and trophectoderm,and the occurrence of attachment and spreading were evaluated.Embryo development was significantly inhibited in cultures supplementedwith 100 ng/ml EGF compared to the controls (P < 0.001).Development of 4-cell embryos to blastocysts occurred in 25%of the EGF group compared to 85% of controls. Spreading occurredin 20% of 4-cell embryos and 30% of blastocysts treated withEGF, compared to 80 and 90% of corresponding controls. In embryosdeveloping from the 4-cell stage, massive growth of the ICMand inhibition of the trophectoderm occurred, whereas both ICMand trophectoderm were inhibited by EGF in embryos developingfrom the blastocyst stage. Following spreading, EGF caused massivegrowth of the ICM and regression of the trophectoderm. Our preliminaryresults show that EGF may be involved in the modulation andcontrol of early embryonic growth and differentiation.     

Expression of cystic fibrosis transmembrane conductance regulator during early human embryo development

Formation of the blastocyst is one of the first morphological changes in early embryonic development. Ion transport has been shown to be crucial for blastocoele cavity formation and expansion, although the mechanisms that underlie this process are presently unknown. As a transmembrane Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR) may participate in ion transport and early blastocoele formation. CFTR mRNA was detected throughout preimplantation embryo development and in the unfertilized oocyte. Immunocytochemistry disclosed the presence of CFTR protein from the 8-cell stage, reaching maximum immunoreactivity at early blastocyst stage embryos. Patch clamp electrophysiology of morulae and blastocysts demonstrated typical CFTR Cl(-) channel activities in the apical membrane of trophectoderm cells. Thus CFTR is expressed both at mRNA and protein levels in human morulae and blastocysts, and functions as a cAMP-regulated apical membrane Cl(-) channel. These data suggest that CFTR may contribute to blastocoele formation in the early human embryo.     

Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts

BACKGROUND: Manual puncture of the trophectoderm of human blastocysts with a needle before vitrification increases their survival rate, but the embryos take a long time to re-expand. This study examined whether causing human blastocysts to collapse by manual pipetting before vitrification would allow more rapid re-expansion and improve pregnancy rates. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were placed in cryoprotectant and then artificially shrunk by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. The shrunken embryos were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. The blastocysts were thawed by warming and then dilution in 1 mol/l sucrose. RESULTS: Of 49 expanded vitrified blastocysts, 48 (98%) re-expanded within 3 h after warming. Following transfer (48 blastocysts in 28 cycles), 14 women (50%) became clinically pregnant, and the implantation rate was 33% (16/48). Eight healthy babies have been born in six deliveries, and the other eight pregnancies are ongoing. To date, there have been no spontaneous abortions. CONCLUSIONS: The results suggest that artificial shrinkage with pipetting is a simple and effective technique to assist successful cryopreservation of expanded blastocysts by vitrification.     

骨桥蛋白对小鼠胚泡黏附和扩展的影响及其机制

目的 探讨骨桥蛋白(OPN)对小鼠胚泡黏附、扩展的影响及机制. 方法 采用纤维蛋白铺板微滴培养法培养胚胎,观察并统计重组小鼠OPN(rmOPN)、OPN抗体及精氨酸-甘氨酸-天冬氨酸多肽(RGD)对胚泡的脱带、黏附和扩展的影响;采用24孔板培养胚胎,酶联免疫吸附测定(ELISA)法检测胚泡培养液基质金属蛋白酶2和9(MMP-2、-9)的浓度. 结果不同浓度的OPN组间胚泡的脱带率、黏附率与对照组相比无显著性差异(P>0.05);但1.0 mg/L和10.0 mg/L OPN组可促进小鼠胚泡提前扩展,72h囊胚扩展率与对照组相比差异有显著性,10.0 mg/L组72h的扩展率显著高于OPN 0.1 mg/L组(P<0.05).OPN抗体和RGD均显著抑制胚泡的脱带、黏附和扩展,与对照组相比,差异有显著性,且随着浓度的增加,抑制作用越明显.OPN促进胚泡分泌MMP-2及MMP-9,且存在时间和剂量依赖性. 结论 OPN在体外可通过促进小鼠胚泡的扩展和分泌MMP-2、-9来调节小鼠早期胚泡着床.