Increased survival of preimplantation mouse embryos in medium with recombinant cytokine LIF

We studied the effects of cytokine LIF on in vitro development of 2-cell mouse embryos to the late blastocyst stage. LIF at 10 ng/ml enhanced the blastocyst formation and hatching from zona pellucida. When blastocysts were cultivated in a medium with LIF for a longer time, the trophoblast adhesive properties and proliferative activity were enhanced. In the presence of this cytokine, the trophoblast cells were attached to the substrate surface and fulfill the function of a sublayer for growth of the inner cell mass colonies with a high activity of endogenous alkaline phosphatase. Expression of LIF was detected in the oviduct and uterus epithelial tissues from day 1 until day 4 of pregnancy, thus suggesting its involvement in early development. According to the data of cultivation, cytokine LIF enhanced the adhesive properties and functional activity of the trophoblast cells, which is essential for implantation of blastocysts in the uterus.     

Significance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts in vitro versus in vivo

We investigated the course of mouse blastocyst hatching in vitro after experimental modulation of the hatching process by growth hormone or by laser treatment and compared it to embryos grown in vivo. When embryos were grown in vitro, successful hatching was dependent on blastocyst expansion and was based on a minimum number of embryonic cells. Embryos grown in the presence of growth hormone were more advanced in their development and hatched earlier. When an artificial opening was laser-drilled into the zona pellucida, hatching occurred at lower numbers of embryonic cells. In vivo, escape from the zona pellucida occurred earlier and independent of blastocyst expansion. However, when we isolated in vivo-grown blastocysts with intact zonae that had developed in vivo and then cultured them in vitro, blastocysts started to expand and hatched the following day when a sufficiently high number of embryonic cells was present. Our data show that successful hatching in vitro is dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding. In vivo, the lower number of embryonic cells detected in zona-free blastocysts indicates that the underlying mechanism of zona escape is different, does not depend on blastocyst expansion, and presumably involves lytic factors from the uterus.     

Increased survival of preimplantation mouse embryos in medium with recombinant cytokine LIF

We studied the effects of cytokine LIF on in vitro development of 2-cell mouse embryos to the late blastocyst stage. LIF at 10 ng/ml enhanced the blastocyst formation and hatching from zona pellucida. When blastocysts were cultivated in a medium with LIF for a longer time, the trophoblast adhesive properties and proliferative activity were enhanced. In the presence of this cytokine, the trophoblast cells were attached to the substrate surface and fulfill the function of a sublayer for growth of the inner cell mass colonies with a high activity of endogenous alkaline phosphatase. Expression of LIF was detected in the oviduct and uterus epithelial tissues from day 1 until day 4 of pregnancy, thus suggesting its involvement in early development. According to the data of cultivation, cytokine LIF enhanced the adhesive properties and functional activity of the trophoblast cells, which is essential for implantation of blastocysts in the uterus.     

Differentiation of blastocysts derived from in vitro-fertilized rhesus monkey ova

Six in vitro-fertilized ova were cultured for 10 to 13 days in vitro. All six had formed blastocysts with cavities, five had hatched from the zona pellucida, and one had attached to the substrate. After fixation and preparation for examination by light and electron microscopy, it was determined that all but the youngest blastocysts had developed substantial amounts of syncytial trophoblast, which morphologically resembled the syncytial trophoblast present in the first 2 days of implantation in vivo. One of the smaller blastocysts had developed syncytial trophoblast but had not hatched from the zona. All of the blastocysts showed indications of loss or inadequate development of the inner cell mass constituents, indicating that the culture conditions were suboptimal for these constituents. Apparent C-type virus particles were abundant, budding from the basal surface of the syncytial trophoblast. Because the type of trophoblast formed was that normally associated with epithelial invasion and formation of the trophoblast plate, it is suggested that such blastocysts would be useful for in vitro implantation studies as well as studies of formation of syncytial trophoblast. However, other methods should be developed for study of postimplantation embryo development. It is also noted that the inadequate differentiation of the epiblast and endoderm would not have been apparent without microscopic examination.     

哺乳动物胚胎透明带脱落机制的研究进展

胚胎着床是一个连续的动态过程,其中胚泡从透明带中准时孵出是着床的关键.透明带脱落的机制主要是子宫或(和)胚泡分泌物部分或全部溶解透明带后,胚泡在细胞数量增加及细胞运动的机械压力作用下通过透明带的某一位点孵出.     

Epiblast isolation by a new four stage method (peeling) from whole bovine cloned blastocysts

We have established a new 4 stage epiblast isolation method from whole bovine cloned blastocysts without using immunosurgery. The new “peeling” method consists of dissolution of the zona pellucida (first stage), elimination of mural trophoblast (second stage), isolation of primitive endoderm and epiblast from polar trophoblast (third stage), and isolation of epiblast from primitive endoderm (fourth stage). The bovine cloned blastocyst consists of 4 different types of cells showing abundant alkaline phosphatase activity. The epiblast origin of isolated cells was confirmed by in vitro differentiation of isolated cells to tubulin β3-positive neurons and by embryoid body formation. The bovine cloned blastocyst origin of isolated epiblasts was confirmed by microsatellite analysis and mitochondrial DNA sequencing analysis. This new method might accelerate establishment of somatic cell nuclear transfer derived embryonic stem cell lines from bovine and other mammals.     

The stimulating effect of recombinant cytokine LIF on the mouse blastocysts during implantation

The effect of recombinant LIF cytokine (Leukemia inhibitory factor) on the isolated mouse embryos at the stages of middle and late blastocyst has been investigated. We have demonstrated here that this agent is necessary in vitro at the stage of normal trophoblast formation after the blastocysts hatch from zona pellucida. This cytokine (10 ng/ml) caused intensification of adhesion and proliferative activity of the trophoblast cells. This is important for intercellular interactions with endometrium and for invasion of embryos into the uterus. The recombinant LIF insignificantly influenced cells of the inner cell mass.     

Developmental potential of isolated blastomeres from early murine embryos

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).     

Production of identical twins by bisection of blastocysts in the cow

Day-8 embryos were recovered by a non-surgical method from superovulated crossbred heifers. Normal expanded blastocysts with a distinct inner cell mass and a trophoblast were released from the zona pellucida and bisected along a sagittal plane into two 'half' blastocysts. Each 'half' blastocyst was replaced in an empty zona pellucida and cultured for 2 h in B2 medium. After culture the 'half' blastocysts were directly transferred to recipient heifers via the cervix. From 11 blastocysts, 11 monozygotic 'half' blastocyst pairs were transferred to 11 recipients: 8 recipients became pregnant, 4 carried twins and one delivered a normal calf and an acardiacus amorphus monster consisting of disorganized embryonic tissues. A further 11 'half' blastocysts were transferred as singletons to 11 recipients. Five recipients were apparently pregnant at Day 42. One returned to oestrus at Day 45, 3 were carrying normal fetuses and 1 a pair of normal twin fetuses when slaughtered at Day 128. It is concluded that even after the first irreversible cellular differentiation which occurs at the blastocyst stage it is still possible to produce identical cattle twins by bisection of the Day-8 blastocyst.     

Peptidomic analysis of blastocyst culture medium and the effect of peptide derived from blastocyst culture medium on blastocyst formation and viability

High‐quality in vitro human embryo culture medium can improve the blastocyst formation rate and blastocyst quality and be beneficial for the clinical application of single blastocyst transfer. Mammalian embryos can secrete protein products into the surrounding medium. As a group of bioactive molecules and degraded proteins, peptides have been shown to participate in various biological processes. Using liquid chromatography‐tandem mass spectrometry, we performed comparative peptidomic analysis of human culture medium in blastocyst formation and nonblastocyst‐formation groups. A total of 201 differentially expressed peptides originating from 157 precursor proteins were identified. Among these, a peptide derived from HERC2 (peptide derived from blastocyst culture medium [PDBCM]) passed through the zona pellucida, was distributed on the perivitelline space, was absent in arrest embryos and highly expressed in high‐quality blastocysts compared with low‐quality blastocysts, and significantly promoted blastocyst formation in a concentration‐dependent manner. These results indicate that PDBCM may be a novel biomarker for predicting blastocyst formation and viability. The mechanism remains unclear and needs to be explored in the future.     

Killing of mouse blastocyst stage embryos by cytotoxic T lymphocytes directed to major histocompatibility complex antigens

A new assay, mixed embryo leukocyte interaction assay, in which the ability of cytotoxic T lymphocytes (CTL) to kill preimplantation mouse embryos could be investigated, is described. CTL were generated both in vitro and in vivo to the H-2b and H-2d haplotypes. The specificity of the CTL was verified by using EL-4 (H-2b) and P815 (H-2d) target cells in a 51Cr-release assay. The cytolytic effect of the CTL on mouse blastocysts was measured by assessing blastocoel retention and inhibition of [3H]thymidine incorporation by the embryos. It was shown that CTL kill blastocyst stage embryos from C57BL/6J (H-2b) and B10.D2 (H-2d) mice with the zona pellucida removed, but not with the zona pellucida intact. These results demonstrate that the H-2 antigens present on mouse blastocysts can be recognized by CTL. It is suggested that one biologic role for the zona pellucida is the prevention of cell-mediated destruction of preimplantation embryos in utero.     

Culture of 5-day horse embryos in microdroplets for 10 to 20 days

Embryos were recovered from the uteri of mares 5 d after ovulation. Six embryos, all morulae, were placed singly in 200-ul droplets of Ham's F-12 with 10% fetal calf serum and cultured at 37 degrees C in a 5% CO(2) atmosphere. The embryos expanded to form blastocysts by the third day of culture. The blastocysts hatched from their zona pellucida, rather than the zona thinning and flaking off, as occurs in vivo. Hatching from the zona pellucida began on the third day of culture and was complete in five of six embryos by the sixth day. The embryonic capsule, normally present in equine embryos after Day 6, was not seen in the cultured embryos. The blastocysts continued to expand until 15 to 17 d of age (10 to 12 d in culture), reaching an average diameter (+/- SD) of 2052 +/- 290 um, after which time they either collapsed or contracted. These results demonstrate that equine embryos can be maintained in long-term culture in vitro, exhibiting continued growth and expansion in the absence of the embryonic capsule.     

Chlorpromazine inhibits the mitotic index, cell number, and formation of mouse blastocysts, and delays implantation of CBA mouse embryos

Chlorpromazine, administered to pregnant CBA mice 56 h after copulation in single doses of 10 or 15 mg/kg bodyweight, inhibited the compaction of embryos, formation of blastocysts, and reduced the mitotic index and cell number of embryos 86 h after copulation but did not adversely influence their viability or induce structural chromosomal aberrations. Blastocyst formation was more severely affected than embryo compaction. When 86-h embryos were treated with chlorpromazine (10 or 15 mg/kg) and subsequently cultured for 120 h, there was delayed hatching from the zona pellucida, delayed attachment to the culture dish, outgrowth of the trophoblast and expansion of the inner cell mass. Mice treated identically and evaluated on the 18th day of gestation had fewer implanted embryos than did controls, and the fetuses weighed less. No resorptions, malformations or significant differences in intrauterine deaths were found. Chlorpromazine given in the same manner but at 0.5 mg/kg did not affect any of the aforementioned criteria. When 56 h embryos were cultured in vitro in the presence of 50 microM-chlorpromazine for a further 40 h, embryo compaction, blastocyst formation, the mitotic index and the total cell number were significantly reduced compared with controls. Blastocyst formation was again more severely affected than embryo compaction. The inhibition of embryo compaction, blastocyst formation, and reduction in mitotic index and cell number associated in this study with chlorpromazine in vivo and in vitro indicate that the drug inhibits the development of cleavage-stage embryos in the mouse. These effects might be mediated by antagonistic effects of calmodulin.     

Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos

Tunicamycin, an antimetabolite which inhibits the N-glycosylation of proteins, does not block the initial cleavages of mouse embryos, even at relatively high concentrations. However, it can interfere with compaction and blastocyst formation. Although tunicamycin treatment from the two-cell or eight-cell stage can cause developmental arrest prior to hatching from the zona pellucida, much higher (sublethal) concentrations of the antimetabolite added at the morula or blastocyst stage do not specifically affect hatching of blastocysts, their attachment to the substratum, or outgrowth of trophoblast cells. The consequence of continuous exposure of embryos to moderate amounts (0.05 to 0.1 μg/ml) of tunicamycin through peri-implantation stages is death of trophoblast cells with little effect upon the cells of the inner cell mass (ICM). The latter give rise to apparently normal early endoderm cells in the presence of the antimetabolite. The incorporation of leucine, mannose, and fucose into acid-insoluble material by ICM cells is only minimally inhibited by tunicamycin. On the other hand, the antimetabolite causes a severe inhibition of incorporation of not only mannose, but also leucine, into acid-insoluble material in trophoblast cells. Thus, trophoblast cells resemble transformed cells by their extreme sensitivity to tunicamycin.     

Blastocyst development after intergeneric nuclear transfer of mountain bongo antelope somatic cells into bovine oocytes

Intergeneric embryos were constructed by nuclear transfer using Mountain Bongo antelope somatic cells fused with enucleated bovine oocytes and their subsequent development in vitro was investigated. After two to six passages, starved or non-starved skin fibroblast cells were used as donor nuclei. In vitro matured bovine oocytes were enucleated by squeezing the first polar body and surrounding cytoplasm through a slit in the zona pellucida. After injection of a somatic cell into the perivitelline space, couplets were fused electrically and activated chemically, then subjected to different embryo culture treatments. Serum starvation had no effect on the frequency of cleavage to two cells or on development to the blastocyst stage in either sequential hamster embryo culture medium (HECM)-6/TCM-199 + serum or HECM-9/TC-199 + serum, or modified synthetic oviduct fluid (mSOF) culture medium. When couplets from non-starved donor nuclei were cultured, the frequency of cleavage (66 +/- 8% vs. 44 +/- 5%), development to >/=9 cells (46 +/- 6% vs. 24 +/- 4%), and formation of blastocysts (24 +/- 5% vs. 11 +/- 2%) were all significantly higher (p < 0.05) in the HECM-6 medium than in mSOF medium. In conclusion, bovine oocytes can support blastocyst development after intergeneric fusion with bongo fibroblasts. This technique could potentially be used as an alternative to using scarce bongo oocytes in attempts to propagate these endangered animals.     

Progressive expression of trophoblast-specific genes during formation of mouse trophoblast giant cells in vitro

The expression of a battery of trophoblast-specific mRNAs was studied during trophectoderm development in vivo and in vitro to assess the use of these mRNAs as markers of trophoblast differentiation and to examine lineage relationships between various trophectoderm derivatives. In situ hybridization of sectioned day 6.5–18.5 mouse embryos localized mRNAs for mouse placental lactogens I and II and mouse proliferin (PLF) to trophoblast giant cells and proliferin-related protein mRNA to the spongiotrophoblast and giant cell layers. A fifth marker, cDNA 4311, was found only in spongiotrophoblast. Day 3.5 blastocyst outgrowths and day 7.5 diploid extraembryonic ectoderm (EX) and ectoplacental cone (EPC) were then cultured to produce polyploid giant cells in vitro. Cultures were processed for in situ hybridization after 2, 4, or 6 days. EX and EPC both formed secondary giant cells, which expressed all markers in the same sequence as was observed in vivo, and primary giant cells in blastocyst outgrowths expressed the early giant cell markers PLF and PL-I on days 4 and 6 of culture. EPC progressed through the sequence 2 days ahead of EX, indicating commitment of EPC to giant cell formation. These results suggest that EX, EPC, and primary and secondary giant cells all share in a common pathway of differentiation and that the highly ordered sequence of gene expression characteristic of this pathway occurs similarly in vivo and in vitro. © 1993 Wiley-Liss, Inc.     

Blastocyst implantation in the Chinese hamster (Cricetulus griseus)

Embryonic development of the Chinese hamster (Cricetulus griseus) was studied from the onset of implantation to the formation of the parietal yolk sac placenta. Implantation began on day 6 of pregnancy, when the embryo became fixed to the uterine luminal epithelium. At this time there was no zona pellucida, and microvilli of the trophoblast and uterine epithelium were closely apposed. Stromal cells immediately adjacent to the implantation chamber began to enlarge and accumulate glycogen. By day 7 the mural trophoblast penetrated the luminal epithelium in discrete area. The trophoblast appeared to phagocytize uterine epithelial cells, although epithelium adjoining the points of penetration was normal. In other areas nascent apical protrusions from the uterine epithelium indented the surface of the trophoblast. The epiblast had enlarged and both visceral and parietal endoderm cells were present. The well-developed decidual cells were epithelioid and completely surrounded the implantation chamber. On day 8 the uterine epithelium had disappeared along the mural surface of the embryo. The embryonic cell mass was elongated and filled the yolk sac cavity. Reichert's membrane was well developed. The uterine epithelial basal lamina was largely disrupted, and the trophoblast was in direct contact with decidual cells. Primary and secondary giant trophoblast cells were present and in contact with extravasated maternal blood. The mural trophoblast formed channels in which blood cells were found in close proximity to Reichert's membrane. Decidual cells were in contact with capillary epithelium and in some cases formed part of the vessel wall. Structural changes occurring in the embryo and endometrium during implantation in the Chinese hamster are described for the first time in this report and are compared to those described for some other myomorph rodents.     

Culturing in vitro produced blastocysts in sequential media promotes ES cell derivation

Embryonic stem (ES) cell lines are routinely derived from in vivo produced blastocysts. We investigated the efficiency of ES cells derivation from in vitro produced blastocysts either in monoculture or sequential culture. Zygotes from hybrid F1 B6D2 mice were cultured in vitro to the blastocyst stage in Potassium (K(+)) simplex optimised medium (KSOM) throughout or in KSOM and switched to COOK blastocyst medium on day 3 (KSOM-CBM). Blastocysts were explanted on a feeder layer of mitomycin C-inactivated murine embryonic fibroblasts (MEF) in TX-WES medium for ES cell derivation. Sequential KSOM-CBM resulted in improved blastocyst formation compared to KSOM monoculture. ES cells were obtained from 32.1% of explanted blastocsyts cultured in KSOM-CBM versus 18.4% in KSOM alone. ES cell lines were characterized by morphology, expression of SSEA-1, Oct-4 and alkaline phosphatase activity, and normal karyotype. These results indicate that in vitro culture systems to produce blastocysts can influence the efficiency of ES cell line derivation.     

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (相似文献   

Investigation of the tissue specificity of the lethal yellow (Ay) gene in mouse embryos

The tissue specificity of the lethal yellow mutant was investigated by separation of blastocyst tissues. Embryos from experimental (Ay/ae X Ay/ae) and control (ae/ae X Ay/ae) crosses of the AG/CamPa inbred strain were recovered at 3.5 days post coitum, cultured for 24 hours, and then mechanically dissected into the component tissues of the blastocyst, the inner cell mass (ICM), and trophectoderm. These fragments were then cultured separately, with or without a feeder layer of inactivated fibroblasts, for an additional 3-5 days. Comparisons between experimental and control crosses indicated that the lethal Ay/Ay embryos were among the blastocysts successfully dissected but that both the ICM and trophectoderm from lethal embryos failed to develop further in vitro, either with or without feeders. With retrospective identification of the lethal embryos, it was found that at 4.5 days, after 1 day of culture, they had formed morphologically normal blastocysts but were frequently more fragile upon dissection and had smaller ICMs. Although none had hatched from the zona pellucida, some had ruptured it and were halfway out. With culture, lethal ICMs showed no development, and lethal trophectoderm usually attached but showed very limited outgrowth. Thus, no rescue of lethal tissue was shown with dissection and in vitro culture, and results are consistent with the gene affecting both tissues of the late blastocyst.