Unbiased contribution of the first two blastomeres to mouse blastocyst development

Several recent studies have proposed a model that the organization of the mouse blastocyst is determined by the pattern of early cleavages: the plane of first cleavage divides the two-cell embryo into embryonic (Em) and abembryonic (Ab) halves, while the timing of the second cleavages specifies which blastomere becomes the Em half. This model is still controversial because of conflicting observations in various studies. Here, we investigated the possibility that the difference between mouse strains contributed to the discrepancy of the findings of different experiments regarding the relationship between the first two cleavages and the blastocyst axial pattern. First, we showed by using a lipophilic, fluorescent tracer that the plane of the first cleavage bears no consistent spatial relationship to the Em-Ab axis of the blastocyst regardless of the genotypic background. Secondly, the order of the second cleavage does not correlate with the Em-Ab polarity of the blastocyst. This was demonstrated by tracing the lineage of the early- and later-dividing two-cell stage blastomeres in the whole embryo as well as by comparing the developmental potential of isolated early- and later-dividing blastomeres and chimeras made entirely of early- or later-dividing blastomeres. These results suggest that contrary to recent studies, the differences between the early- and later-dividing blastomeres of the two-cell embryo are not functionally evident and do not define the Em-Ab polarity of the blastocyst. The significance of these findings is discussed in relation to human assisted reproduction and preimplantation genetic diagnosis.     

Paracrine effects of bFGF and KGF on the process of mouse blastocyst implantation

Implantation is a complex process that requires the interaction of the blastocyst, and subsequently, that of the developing embryos with the endometrium. Several growth factors and cytokines are involved in implantation, but the details of their actions as related to the regulation of blastocyst implantation remain unclear. In the present study, the RT-PCR method was used to determine the gene expression of basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1 (FGFR1), FGF receptor 2 (FGFR2), and KGF receptor (KGFR) in mouse embryos and in the stromal and epithelial cells of the uterine endometrium. Basic FGF and KGF mRNA were expressed in the endometrial cells, but were not expressed in the embryos. The mRNAs of receptors for bFGF and KGF were expressed in the blastocysts and in the in vitro implanting embryos, suggesting that bFGF and KGF may exert paracrine effects on blastocyst implantation. In this mouse model of blastocyst implantation, it was found that transforming growth factor α (TGF-α) at the concentrations of 1 ng/ml and 10 ng/ml significantly enhanced the blastocyst attachment and trophoblast spreading and increased trophoblast surface area. Relatively high concentrations of bFGF (100–500 ng/ml) significantly enhanced the rates of blastocyst attachment and of trophoblast spreading and promoted the expansion of the surface area of the implanting embryos. Unlike the rates of blastocyst attachment and trophoblast spreading, the surface area of the spreading embryos was significantly increased by addition of KGF (1–100 ng/ml). These results suggest that the bFGF and KGF derived from the endometrial cells exert paracrine effects on the process of implantation by stimulating trophoblast outgrowth through their cognate receptors. Mol. Reprod. Dev. 50:54–62, 1998. © 1998 Wiley-Liss, Inc.     

A simplified method for mouse blastocyst injection

A simplified method for blastocyst injection is described. Two instruments only are used: one micropipette for holding the blastocyst and another for injection. Both instruments are easily made by cutting a pulled capillary. No bevelling or polishing is needed. The instruments may be used for many successive injections.     

In vitro development of core cells of the inner cell mass of the mouse blastocyst: effects of conditioned medium.

Blastocysts submitted to two rounds of immunosurgery give rise to cores of presumptive ectoderm cells, many of which do not survive for more than 48 hours when cultured individually. Precoating of the culture plates with conditioned medium (CM) from PYS-2 cells increases the incidence with which cores regenerate an outer layer. This procedure also improves the survival frequency of the cores, but only for a limited period of time. The small number of cores which survive for two weeks or more, either in uncoated or CM-coated plates, give rise to any array of cell types, including giant cells resembling trophoblast.     

The cell biology of blastocyst development.

Preimplantation development encompasses the "free"-living period of mammalian embryogenesis, which culminates in the formation of a fluid-filled structure, the blastocyst. Cavitation (blastocyst formation) is accompanied by the expression of a novel set of gene products that contribute directly to the attainment of cell polarity with the trophectoderm, which is both the first epithelium of development and the outer cell layer encircling the inner cell mass of the blastocyst. Several of these gene products have been identified and include the tight junction (ZO-1), Na/K-ATPase (alpha and beta subunits), uvomorulin, gap junction (connexin43), and growth factors such as transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF). This review will examine the role(s) of each of these gene products during the onset and progression of blastocyst formation. The trophectodermal tight junctional permeability seal regulates the leakage of blastocoel fluid and also assists in the maintenance of a polarized Na/K-ATPase distribution to the basolateral plasma membrane domain of the mural trophectoderm. The polarized distribution of the Na/K-ATPase plays an integral role in the establishment of a trans-trophectoderm Na+ gradient, which drives the osmotic accumulation of water across the epithelium into the nascent blastocoelic cavity. The cell adhesion provided by uvomorulin is necessary for the establishment of the tight junctional seal, as well as the maintenance of the polarized Na/K-ATPase distribution. Growth factors such as TGF-alpha and EGF stimulate an increase in the rate of blastocoel expansion, which could, in part, be mediated by secondary messengers that result in an increase in Na/K-ATPase activity. Insight into the mechanism of cavitation has, therefore, directly linked blastocyst formation to trophectoderm cell differentiation, which arises through fundamental cell biological processes that are directly involved in the attainment of epithelial cell polarity.     

Ultrastructure of blastocyst attachment in the mouse

Summary Mouse embryos have been examined with light and electron microscopy after fixation by perfusion with glutar aldehyde, and embedding in plastic.The Zona pellucida is dissolved gradually around the blastocyst just prior to attachment, and Zona free blastocysts exist only for a very short time.Blastocyst attachment is established when the trophoblast and uterine cell surface membranes lie within 150 Å apart over wide areas. The uterine epithelium does not show any signs of degeneration.Trophoblast attachment probably precedes decidual cell reaction.This work was supported by the Swedish Government Funds for Supporting Medical Research and the Swedish Medical Research Council (Project No. 12 X-70-02).     

Cell surface changes on the mouse blastocyst at implantation

Cell surface changes on the trophectoderm of the mouse blastocyst have been followed in the periimplantation period using electronhistochemical techniques. Examination of the ability of the trophectoderm to bind positively charged colloidal iron particles before and after enzyme treatment has shown that sialic acid-containing glycoproteins make a considerable contribution to the negative charge on the blastocyst surface. At implantation these membrane components are lost or undergo modification independently of direct maternal influence as indicated by a marked decline in colloidal iron binding at this time, both in vivo and in vitro. The findings are discussed in relation to other surface changes on the blastocyst and to the initiation of implantation.     

The analysis of chromatin organisation allows selection of mouse antral oocytes competent for development to blastocyst

Mouse antral oocytes can be classified in two different types termed SN or NSN oocytes, depending on the presence or absence, respectively, of a ring of Hoechst 33342-positive chromatin surrounding the nucleolus. The aim of the present study was to test the developmental competence to blastocyst of the two types of oocytes. Here we show that following isolation, classification and culture of cumulus-free antral oocytes, 14.7% and 74.5% of NSN and SN oocytes, respectively, reached the metaphase II stage. When fertilised and further cultured none of the metaphase II NSN oocytes developed beyond the 2-cell stage whilst 47.4% of the metaphase II SN oocytes reached the 4-cell stage and 18.4% developed to blastocyst. The findings reported in this paper may contribute to improved procedures of female gamete selection for in vitro fertilisation of humans and farm animals. Furthermore, the selection of oocytes with better developmental potential may be of interest for studies on nuclear/cytoplasm interaction, particularly in nuclear-transfer experiments.     

Closed embryo culture system improved embryological and clinical outcome for single vitrified-warmed blastocyst transfer: A single-center large cohort study

While some studies have shown that the closed embryo culture system (CCS) is a possible improvement over standard embryo culture systems (STS) in terms of early embryonic development, information on clinical outcomes of culturing blastocysts following single vitrified-warmed blastocyst transfer (SVBT) in the CCS and STS remains limited. Therefore, the objective of this single-center, large-cohort, retrospective study was to compare embryonic development until the blastocyst stage and clinical outcomes following SVBT between CCS and STS. From May 2017 to October 2018, 2420 oocytes from 1402 patients who underwent in vitro fertilization and blastocyst culture after minimal stimulation were divided into two groups (CCS and STS). The main outcome measures in the two groups were embryological (blastocyst formation rates and utilized blastocyst rates) and clinical outcomes (ongoing pregnancy rates) after a single vitrified-warmed blastocyst transfer (SVBT). There were no significant differences in the blastocyst formation rates between the CCS and STS groups (59.6% versus 59.1%, p = 0.81). However, there were significant differences in utilized blastocyst rates (51.0% versus 46.6%, p < 0.05). Ongoing pregnancy rates per SVBT cycle were significantly higher in the CCS group than in the STS group (41.4% versus 34.4%, p < 0.05). Moreover, after applying multivariable logistic regression analysis, the type of embryo culture system (CCS to STS, adjusted odds ratios: 1.41, 95% CI: 1.04–1.91) was correlated with ongoing pregnancy. Our study suggests that compared to STS, CCS could improve utilized blastocyst rates and ongoing pregnancy rates to a greater extent, following SVBT.     

Protein degradation in the mouse blastocyst.

The degradation characteristics of 56 individual newly synthesized proteins of the Day 4 mouse blastocyst have been examined employing double isotope labeling of proteins for half-life measurement and two-dimensional electrophoresis for separation of proteins. The half-lives ranged from 1 to approximately 30 h with a mean of 12.4 h. Several proteins appeared to have half-lives greater than 30 h but decay times were insufficient to provide precise information for these proteins. The results suggest there is a tendency for proteins with acidic isoelectric points to be degraded more rapidly than basic proteins, and for high molecular weight proteins to be degraded more rapidly than low molecular weight proteins. Although the regressions of these two parameters on half-life were not significant, the direction and magnitude of the trends were similar to those previously described for liver proteins. Two specific proteins, tubulin and actin, were tentatively identified, and their half-lives determined. Tubulin had a half-life of 9.0 h. The half-lives of the provisionally identified gamma, beta, and alpha forms of actin were 2.2, 8.7, and 5.4 h respectively.     

Imaging filopodia dynamics in the mouse blastocyst

During mammalian development, the first cell lineage diversification event occurs in the blastocyst, when the trophectoderm (TE) and the inner cell mass (ICM) become established. Part of the TE (polar) remains in contact with the ICM and differs from the mural TE (mTE) which is separated from the ICM by a cavity known as the blastocoele. The presence of filopodia connecting ICM cells with the distant mural TE cells through the blastocoelic fluid was investigated in this work. We describe two types of actin-based cell projections found in freshly dissected and in vitro cultured expanding blastocysts: abundant short filopodia projecting into the blastocoelic cavity that present a continuous undulating behavior; and long, thin traversing filopodia connecting the mural TE with the ICM. Videomicroscopy analyses revealed the presence of vesicle-like structures moving along traversing filopodia and dynamic cytoskeletal rearrangements. These observations, together with immunolocalization of the FGFR2 and the ErbB3 receptors to these cell extensions, suggest that they display signal transduction activity. We propose that traversing filopodia are employed by mitotic mTE cells to receive the required signals for cell division after they become distant to the ICM.     

Oct4 protein remains in trophectoderm until late stages of mouse blastocyst development

Oct-4, the marker of pluripotent cells, is crucial for murine preimplantation development. During the formation of the blastocyst Oct-4 is downregulated in the trophectoderm (TE) and its expression becomes restricted to the inner cell mass (ICM). In order to determine the exact timing of the disappearance of Oct-4 protein from TE we analyzed the localization and level of Oct-4 at different stages of blastocyst development. The presence of Oct-4 protein was determined by immunohistochemistry using confocal microscopy. We found that the downregulation of Oct-4 protein in TE of mouse blastocysts progresses gradually during development, and Oct-4 protein persists in some of the TE cells at least until the expanded blastocyst (120-140 cells) stage. Our findings indicate that the switching-off of the Oct-4 expression is not necessary for the trophectoderm formation. The complete elimination of Oct-4 protein from TE occurs at the period of blastocyst implantation, when lack of Oct-4 is required for the proper functioning of the trophectoderm.     

A comparative study of mouse liver and mouse blastocyst DNA-dependent RNA polymerases.

DNA-dependent RNA polymerase has been studied in adult mouse liver and mouse blastocysts. The enzyme from mouse liver was resolved into three enzyme forms by DEAE-Sephadex chromatography. Two of the forms, IA and IB, are insensitive to α-amanitin, have low $\text{Mn}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ activity ratios, and are optimally active at low ionic strength. Form II is inhibited by α-amanitin, has a higher $\text{Mn}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ activity ratio, and is most active at high ionic strength. An optimal reaction temperature of 37 ° C was found for all enzyme forms. All of the isolated enzyme forms are inhibited by the exotoxin from Bacillus thuringiensis and the inhibition can be partially reversed by increased ATP levels. Forms IA and IB are most active with native template while form II prefers denatured DNA.The blastocyst RNA polymerase activity exhibits similar requirements for divalent metal ions and ionic strength to the purified liver enzymes. The maximum inhibition of blastocyst RNA polymerase obtained with α-amanitin and exotoxin differs from that observed for purified liver enzymes but is similar to the inhibition of liver homogenate. However, the concentrations of inhibitor required for maximum inhibition by α-amanitin and exotoxin is different for the blastocyst and liver homogenate enzymes.     

Effects of asynchrony on rabbit blastocyst development

The development of synchronously and asynchronously transferred rabbit morulae (recovered at Day 3 p.c.) and blastocysts (recovered at Day 4 p.c.) was investigated before the anticipated time of implantation. The results obtained with various techniques (evaluation of gross morphology, measurement of diameter, thymidine incorporation, light and electron microscopy) led basically to the same conclusions. Embryos being asynchronously transferred to the uterus of recipient rabbits survived, at least in terms of certain cellular functions like cell proliferation, for more than 2 days. Development, however, was clearly retarded and ultrastructural examination revealed substantial cell damage. Some blastocysts showed, even after 3 days, normal growth and cell proliferation indicating considerable differences between individuals in the ability to compensate for suboptimal developmental conditions before implantation. In general, this ability was greater in the transferred Day-3 morulae than in the Day-4 blastocysts. Embryonic growth and the ability to dissolve the zona pellucida, to synthesize crystalloid bodies and to differentiate extraembryonic endoderm indicated the maintenance of some developmental functions under asynchronous conditions. Blastocyst development was influenced by the progestational stage of the recipient. At 1 day after transfer into asynchronous older uteri, blastocyst diameters were larger and cell proliferation was increased compared with all other groups, suggesting an attempt of the blastocyst to adjust to the more advanced maternal milieu. Development in asynchronous younger uteri was delayed. No comparable differences in development were found in cultured embryos for which the media had been supplemented with flushings from the same progestational uterine stages as used for transfer. Thymidine incorporation in cultured embryos did not differ between the various supplements (P greater than 0.05) and was generally lower than in chronologically aged asynchronously transferred embryos (P less than 0.05 for Day-3 and P less than 0.001 or P greater than 0.05 for Day-4 embryos).     

The effects of L- and D-carnitine administration on cardiovascular development of the chick embryo

A single 1.0-ml volume of L- or D-carnitine solution, at several selected mmole concentrations, was applied to the extraembryonic membranes of 3- and 4-day chick embryos in ovo. Hamburger-Hamilton stages of chick development ranged from 17 to 23. During the 17-18th days of incubation, embryos were dissected, and both survival and intracardiac anomaly rates were determined. Only at extremely high doses, both stereoisomers of carnitine exhibited a statistically significant toxigenic effect (p less than 0.001) as measured by a sharp decrease in survival rate when compared to chick Ringer's saline controls. Furthermore, since the anomaly rates became significant only near the LD50's, this indicated that intracardiac anomalies were induced only at toxic doses. Therefore, it is suggested that cardiovascular teratogenicity may be the result of toxicity. Below the LD50, anomaly rates were not significantly different from those of control embryos. In comparison, L- and D-carnitine were significantly different from one another (p less than 0.001) both in survival rate and percent affected embryos at a dose of 0.5 mmole. In summary, exogenous carnitine administration to the chick embryo does not appear to be deleterious to the developing cardiovascular system.     

The effect of clomiphene citrate on blastocyst development and implantation in the rabbit

Summary The induction of ovulation with clomiphene citrate (CC) in human patients results in a high ovulation rate but achieves a relatively low pregnancy rate. To clarify the possible role of CC in interfering with the normal reproductive physiology and embryology, we have used our rabbit model and transferred 4-day-old blastocysts from untreated donors to CC-treated pseudopregnant recipients and from CC-treated donors to untreated pseudopregnant recipients to study embryonic development and implantation. Each group was further subdivided into two subgroups, one receiving CC before and the other after ovulation. CC was administered subcutaneously in three consecutive doses of 10 mg/kg body weight. Ovulation was induced with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG).The implantation rate of the control group, evaluated on day 8 of pregnancy, reached 62.0%. When recipients were treated with CC before ovulation, implantation rate was reduced to 18.8% (P < 0.0002), and to 20.0% (P < 0.003) when CC was administered after ovulation. The implantation rate of blastocysts transferred from donors, treated before ovulation, is 22.2% (P < 0.0055), however, reached 70.8% when treatment was started after ovulation. All implantations were analysed microscopically and showed normal morphological features.Our results demonstrate a potential multiple effect of CC, first on the endometrium by altering its receptivity for the implanting conceptus, second, on tubal physiology by altering egg transport, and finally on ovum maturation before ovulation interfering with development of blastocysts. These parameters may all result in rapid decrease in establishment of implantations and in turn in very low pregnancy rates.     

Effect of delaying DNA replication on blastocyst formation in the mouse

Abstract. Differentiation in the early mouse embryo involves cellular responses to both spatial and temporal signals. The temporal signals that trigger blastocyst formation, the first differentiative event, are not yet understood, but it has been suggested that the numbers of DNA replications undergone since fertilization might act as the timing mechanism. Preimplantation mouse embryos were treated with aphidicolin, an inhibitor of eukaryotic DNA polymerase a, for eight hours during the S phase of the fourth cleavage division. This treatment produced a delay in cell division but the morphologic event of cavitation, which signals the onset of blastocyst formation, was not delayed. Treated embryos actually cavitated a few hours ahead of control embryos at approximately half the cell number. This result indicates that blastocyst formation is not timed by the number of DNA replicative cycles completed since fertilization, but by some other intrinsic cellular clock.     

Trophectoderm cell subpopulations in the periimplantation mouse blastocyst

Trophectoderm of the preimplantation mouse blastocyst is composed of two cell subpopulations relative to their proximity to the inner cell mass. The polar trophectoderm overlying the inner cell mass proliferates to form the ectoplacental cone, and the mural trophectoderm endoreplicates and gives rise to giant cells. We examined specific differences in the two trophectoderm cell populations using a lectin (Dolichos biflorus) to detect cell surface characteristics and a simple sugar (D-Gal) to detect differences in incorporation. During the first day of delayed implantation, the mural trophectoderm presented twice as many lectin binding sites as did the polar trophectoderm. The mural trophectoderm of both nondelaying and delayed implantation blastocysts showed a greater rate of incorporation of the tritiated sugar by presenting more reduced silver grains in radioautograms. These results indicate that the mural trophectoderm and polar trophectoderm are two distinct cell types in the periimplantation blastocyst.