Fertilization and early embryolgoy: Lineage tracing demonstrates that blastomeres of early cleavage-stage human pre-embryos contribute to both trophectoderm and inner cell mass

We injected a fluorescent lineage tracer (Texas Red-lysinedextran)into individual blastomeres of donated human diploid 2- to 8-cellpre-embryos and cultured them to blastocysts. Once pre-embryosreached the expanded blastocyst stage, they were fixed and examinedin a scanning confocal microscope to identify the location offluorescent tracer. In successfully injected pre-embryos thatdeveloped to expanded blastocysts, we found that randomly injectedblastomeres formed both trophectoderm (TE) and inner cell mass(ICM). More labelled progeny were found in TE than in ICM. Ourresults show that individual early blastomeres are not yet committedto form either TE or ICM but instead can form both rudiments.     

Polarity of the mouse embryo is established at blastocyst and is not prepatterned

Polarity formation in mammalian preimplantation embryos has long been a subject of controversy. Mammalian embryos are highly regulative, which has led to the conclusion that polarity specification does not exist until the blastocyst stage; however, some recent reports have now suggested polarity predetermination in the egg. Our recent time-lapse recordings have demonstrated that the first cleavage plane is not predetermined in the mouse egg. Here we show that, in contrast to previous claims, two-cell blastomeres do not differ and their precise future contribution to the inner cell mass and/or the trophectoderm cannot be anticipated. Thus, all evidence so far strongly suggests the absence of predetermined axes in the mouse egg. We observe that the ellipsoidal zona pellucida exerts mechanical pressure and space constraints as the coalescing multiple cavities are restricted to one end of the long axis of the blastocyst. We propose that these mechanical cues, in conjunction with the epithelial seal in the outer cell layer, lead to specification of the embryonic-abembryonic axis, thus establishing first polarity in the mouse embryo.     

Birth of a healthy infant following trophectoderm biopsy from blastocysts for PGD of beta-thalassaemia major

PGD is a well accepted reproductive choice for couples at genetic risk and involves the diagnosis and transfer of unaffected IVF embryos. PGD for monogenetic diseases is most commonly accomplished by the biopsy of one or two blastomeres from cleavage stage embryos, followed by PCR-based protocols. However, PCR-based DNA analysis of one or two cells is subject to several problems, including total PCR failure, or failure of one allele to amplify. Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development. The aim of this study was to develop a safe, reliable technique for the biopsy of trophectoderm cells from human blastocysts. This case report demonstrates that removal of trophectoderm cells prior to blastocyst transfer is compatible with implantation and development to term. Here we report successful PGD for beta-thalassaemia following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant.     

Stereological study of mouse uterine and in vitro grown blastocysts: Cell numbers and volumes

Mouse blastocysts were studied to determine if there were differences in cell number and volumes between those that were (1) derived from the uterus prior to implantation on the afternoon of day 4 of pregnancy and (2) those that were cultured for 72 hr from two-cell-stage embryos. Blastocysts were fixed, embedded in resin, and serially sectioned at 1.5 or 2 μm. Photographic prints of alternate sections were used to count the numbers of inner cell mass (ICM) and trophectoderm cells. Cavalieri's direct estimator was applied to the same prints to estimate the volume of the whole blastocyst. Point counting was used to determine the volumes of the ICM, trophectoderm, and zona pellucida. The number of cells and size of the ICM were similar between the two groups of blastocysts, although it was found that the ICM of uterine embryos that did not have a zona pellucida were smaller than the ICM of those that did. There were twice as many trophectoderm cells in the blastocysts that were cultured from two-cell embryos, and these cells were also found to be larger. Furthermore, the volume of the zona pellucida was less in the uterine blastocysts. This study indicates that, while trophectoderm proliferation is enhanced in vitro, the ICM is more constant and thus may be self-regulating and independent of the growth conditions of the blastocyst as a whole. This study also suggests partial zona lysis occurs in utero and occurs either at a reduced rate or not at all in vitro. © 1993 Wiley-Liss, Inc.     

Ectopic expression of Fgf3 leads to aberrant lineage segregation in the mouse parthenote preimplantation embryos

Background: Parthenogenetic mammalian embryos were reported to die in utero no later than the 25‐somite stage due to abnormal development of both embryonic and extraembryonic lineages. Interestingly, it has been shown that parthenogenetic ICM cells tend to differentiate more into primitive endoderm cells and less into epiblast and ES cells. Hence we are interested in studying the molecular mechanisms underlying lineage defects of parthenotes. Results: We found that parthenote inner cell masses (ICMs) contained decreased numbers of Sox2⁺/Nanog⁺ epiblast cells but increased numbers of Gata4⁺ primitive endoderm cells, indicating an unusual lineage segregation. We demonstrate for the first time that the increased Gata4 level in parthenotes may be explained by the strong up‐regulation of Fgf3 and Fgfr2 phosphorylation. Inhibition of Fgfr2 activation by SU5402 in parthenotes restored normal Nanog and Gata4 levels without affecting Fgf3, indicating that Fgf3 is upstream of Fgfr2 activation. In parthenote trophectoderm, we detected normal Cdx2 but ectopic Gata4 expression and reduced Elf5 and Tbr2(Eomes) levels. Conclusions: Taken together, our work provides for the first time the insight into the molecular mechanisms of the developmental defects of parthenogenetic embryos in both the trophectoderm and ICM. Developmental Dynamics 241:1651–1664, 2012. © 2012 Wiley Periodicals,Inc.     

Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells

The histone H3 Lys 9 (H3K9) methyltransferase Eset is an epigenetic regulator critical for the development of the inner cell mass (ICM). Although ICM-derived embryonic stem (ES) cells are normally unable to contribute to the trophectoderm (TE) in blastocysts, we find that depletion of Eset by shRNAs leads to differentiation with the formation of trophoblast-like cells and induction of trophoblast-associated gene expression. Using chromatin immmunoprecipitation (ChIP) and sequencing (ChIP-seq) analyses, we identified Eset target genes with Eset-dependent H3K9 trimethylation. We confirmed that genes that are preferentially expressed in the TE (Tcfap2a and Cdx2) are bound and repressed by Eset. Single-cell PCR analysis shows that the expression of Cdx2 and Tcfap2a is also induced in Eset-depleted morula cells. Importantly, Eset-depleted cells can incorporate into the TE of a blastocyst and, subsequently, placental tissues. Coimmunoprecipitation and ChIP assays further demonstrate that Eset interacts with Oct4, which in turn recruits Eset to silence these trophoblast-associated genes. Our results suggest that Eset restricts the extraembryonic trophoblast lineage potential of pluripotent cells and links an epigenetic regulator to key cell fate decision through a pluripotency factor.     

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.     

Cellular characterization of blastocysts derived from rabbit 4-, 8- and 16-cell embryos and isolated blastomeres cultured in vitro

The purpose of this study was to investigate the developmental potential of isolated rabbit blastomeres under various culture conditions to gain insight into their ability to form the two cell lineages of a viable blastocyst. Intact embryos at the 4-cell, 8-cell, 16-cell stages and blastomeres isolated from 4-, 8- and 16-cell rabbit embryos (1/4, 1/8 or 1/16 blastomeres respectively) were cultured in drops of one of three different media, each supplemented with either fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA). The effects of the extracellular matrix fibronectin (FN) on the development of isolated rabbit blastomeres were also investigated. Supplementation of the medium with FCS yielded a higher (P < 0.05) proportion of blastocysts than BSA or PVA, predominantly from 1/4 blastomeres. No major differences were found between the three basic culture media. In 1/4, 1/8 or 1/16 blastomeres, blastocyst formation rates were greater (P < 0.05) in groups cultured in matrix-free (54.5, 59.6 and 54.6% respectively) than in FN-coated groups (35.4, 46.0 and 26.1% respectively). Only in blastocysts derived from 1/4 blastomeres, were the numbers of inner cell mass (ICM) and total cells of blastocysts higher (P < 0.05) in FN-coated groups than in matrix-free groups (12.7 +/- 1.1 versus 8.5 +/- 0.7 ICM, 73.8 +/- 3. 7 versus 57.8 +/- 3.3 total cells). The percentage of blastocysts derived from single blastomeres with ICM cells decreased with increasing cell stage of the parent embryos in FN-coated (93.6, 78.3 and 44.0%, respectively) as well as matrix-free groups (96.2, 69.3 and 55.2%). In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, approximately 20-30% of blastomeres did not develop into normal blastocysts but formed sheets with 30-50 cells attached to the bottom of the dishes. These results indicate that the development of rabbit blastomeres shares important characteristics with those from mouse and domestic species and may thus aid in developing an efficient culture system for blastomeres, derived from human embryos.     

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.     

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.     

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.     

An investigation of the fate of cells transplanted orthotopically between morulae/nascent blastocysts in the mouse

A technique has been devised for selectively replacing someinside cells (ICs) or outside cells (OCs) of late morulae/nascentblastocysts with corresponding cells from genetically dissimilar,synchronous embryos. The main purpose was to determine whetherthe inner cell mass (ICM) contributes cells to the overlyingpolar trophectoderm at any stage during the blastocyst phaseof development. Notwithstanding the high incidence and levelof chimaerism in ICM derivatives of postimplantation conceptusesobtained in the IC transplantation experiments, trophoblasttissue was composed entirely of host cells in the majority ofcases. Even where a donor IC contribution to trophoblast wasdetected there were strong grounds for suspecting that it wasdue to tissue contamination rather than genuine chimaerism.Thus, not only did such contributions differ in both level anddistribution from those produced by transplantation of OCs butthey also varied markedly in frequency according to the dayof gestation on which conceptuses were dissected. The possibilitythat ICs regularly colonize the polar trophectoderm but failto persist there was excluded by the results of short-term transplantation experiments using an in-situ genetic marker. These findingsoffer no support for the hypothesis that the ICM serves as apopulation of stem cells for the trophectoderm as well as theprimitive endoderm and ectoderm during normal development. Thefrequency of chimaerism was lower in OC than IC transplantationexperiments. Nevertheless, in a substantial proportion of chimaeras,OCs colonized derivatives of the ICM. This is consistent withevidence from other studies that some outside cells divide differentiallyat 5th cleavage to produce an OC plus an IC rather than twoOCs.     

Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage

Normally fertilized human embryos biopsied 3 days after in-vitro fertilization (IVF) have been examined for effects on viability and development in vitro after removal of one or two cells at the 8-cell stage (1/8 and 2/8) from each embryo. A high proportion of 7/8 and 6/8 biopsied and unmanipulated embryos developed to the blastocyst stage between days 5 and 6 (79, 71 and 59%, respectively), and many biopsied embryos (56%) hatched from the zona pellucida in vitro. The viability of biopsied embryos which developed to the blastocyst stage was assessed by daily non-invasive measurement of the uptake of two energy substrates, glucose and pyruvate. Uptake of both substrates was generally lower in 7/8 and 6/8 biopsied embryos but only in proportion to the reduced cellular mass. The total cell number and the numbers of both trophectoderm (TE) and inner cell mass (ICM) cells in biopsied embryos at the blastocyst stage, counted by differential labelling of their nuclei, were also reduced in proportion but the ratio of ICM to TE cells was maintained in both 7/8 and 6/8 biopsied embryos. We conclude that removal of one or two cells at the 8-cell stage, while reducing the cellular mass, does not adversely affect the preimplantation/development of biopsied embryos in vitro and suggest that this approach could be used for preimplantation diagnosis of genetic defects.     

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.