Early Cleavage Stage Embryos Research Articles

Maternal beta-catenin and E-cadherin in mouse development.

The oocyte to embryo transition in metazoans depends on maternal proteins and transcripts to ensure the successful initiation of development, and the correct and timely activation of the embryonic genome. We conditionally eliminated the maternal gene encoding the cell adhesion molecule E-cadherin and partially eliminated the beta-catenin gene from the mouse oocyte. Oocytes lacking E-cadherin, or expressing a truncated allele of beta-catenin without the N-terminal part of the protein, give rise to embryos whose blastomeres do not adhere. Blastomere adhesion is restored after translation of protein from the wild-type paternal alleles: at the morula stage in embryos lacking maternal E-cadherin, and at the late four-cell stage in embryos expressing truncated beta-catenin. This suggests that adhesion per se is not essential in the early cleavage stage embryos, that embryos develop normally if compaction does not occur until the morula stage, and that the zona pellucida suffices to maintain blastomere proximity. Although maternal E-cadherin is not essential for the completion of the oocyte-to-embryo transition, absence of wild-type beta-catenin in oocytes does statistically compromise developmental success rates. This developmental deficit is alleviated by the simultaneous absence of maternal E-cadherin, suggesting that E-cadherin regulates nuclear beta-catenin availability during embryonic genome activation.

Effects of culture conditions on IVF outcome

Although in vitro fertilization (IVF) success rates have improved over the past decade, multiple pregnancies have become a formidable problem. The solution to this problem seems simple by mandating the reduction in numbers of embryos transferred. However, this is typically not accomplished without a compromise in the pregnancy rate. There have been a number of approaches designed to address high order multiple pregnancies from multi factorial analysis of early cleavage stage embryos to the development of extended culture systems, both of which require manipulations in the culture environment. Manipulations in embryo culture environment may not be benign. Several studies have demonstrated that adverse culture conditions have effects on gene expression and imprinting. Studies have also demonstrated that singleton human IVF babies have lower birth weight and higher incidence of congenital anomalies than natural conception babies. All of these factors need to be considered in relation to long term viability of IVF babies and the Barker hypothesis.

Survival and developmental potential of stored human early cleavage stage embryos

Human early cleavage stage embryos which survive cryopreservation and thawing fully intact demonstrate similar developmental potential to equivalent non frozen embryos when returned to the in vivo environment, whereas blastomere loss is directly related to the loss of potential for subsequent implantation in thawed embryos. This suggests that blastomere lysis during freezing and thawing does not occur preferentially in non viable blastomeres. Prefreeze growth rate rather than prefreeze blastomere number per se correlates with the developmental potential of stored embryos. When blastomere loss occurs as a consequence of cryopreservation, development of thawed early cleavage stage embryos to the blastocyst stage in vitro is impaired and the resultant blastocysts have a reduced total cell content. Blastomere loss is more prevalent in embryos which have been biopsied for preimplantation genetic diagnosis but this increased sensitivity can be circumvented by modification of the standard cryopreservation protocol.

RNAi in cultured Drosophila cells.

Double-stranded RNA (dsRNA)-mediated interference, or RNAi, has emerged as an effective technique to phenocopy the loss of function of a given gene product. With this tool researchers can study the functions of individual molecules in living cells and elucidate the mechanisms that regulate cell division. For example, many molecules that are important for regulating mitosis and for controlling the assembly of the mitotic spindle are mutated in different cancer cell types (for a review, see ref. 1). Functional analysis in vivo of molecules that play a role in mitosis is best implemented by a genetic analysis. For this, genetically malleable organisms such as Drosophila, Caenorhabditis elegans, yeast, and other micro-organisms have been extremely useful. Whereas genetic analysis usually requires a long-term effort, RNAi provides a rapid method for the reverse genetic analysis of gene product function and can be exploited to great advantage. In the era of sequenced genomes, this technique provides a valuable tool for functional genomics. Here, a detailed procedure for RNAi in Drosophila cells in culture is presented. RNA interference was first described using C. elegans (2), although the phenomenon has been described in plants as posttranscriptional gene silencing (PTGS) (3) and as “quelling” in Neurospora (4). Furthermore, RNAi has been demonstrated on a number of organisms (2-9). For Drosophila, RNAi has been accomplished by injection of dsRNA into early syncytial cleavage stage embryos (6). Subsequently, heritable RNAi has been achieved in C. elegans and Drosophila using transgenic dsRNA “hairpin”-generating constructs (10-13). An important advance came when RNAi was demonstrated with cultured Drosophila cells (7). More recently, RNAi has been applied successfully to vertebrate cells in culture using short interfering RNAs (siRNAs) (14,15). For this, the first step in the cellular response mechanism to dsRNA (see below) has to be bypassed, because full-length dsRNAs produce nonspecific effects in vertebrate cells (16-19). RNAi-mediated interference occurs by a posttranscriptional mechanism that targets mRNA homologous to the dsRNA that is introduced for destruction (for reviews, see refs. 20-22). Using Drosophila embryo and S2 cell extracts, the mechanisms for RNAi are being elucidated (16,23). In these extracts, dsRNA is cleaved into 21- to 25-bp siRNAs with 5′ phosphates, 3′ hydroxyl groups, and contain two to three nucleotide 3′ overhangs. dsRNA cleavage is mediated by Dicer, an ATP-dependent RNaseIII family RNase (24). siRNAs assemble into an approx 360-kDa complex called RNA-induced silencing complex (RISC) in Drosophila extracts (25). The siRNAs then unwind in an ATP-dependent manner (23,25). The single-stranded siRNAs in the RISC complex provide the homologous targeting to mRNA (23,26), enabling degradation by the RNase associated with RISC. Furthermore, Argonaute proteins are components of RISC (27) with homologs in plants, fungi, and C. elegans that are required for RNAi in those organisms (28-30). The degraded target mRNA appears to then be cycled into new siRNAs that repeat the process in an RNA polymerase-dependent cycle of mRNA degradation and siRNA production (31). The complete mechanism for RNAi has not been elucidated. A teleological explanation for the existence of a mechanism to destroy mRNAs in response to homologous dsRNA has been proposed (32). It has been suggested that RNAi evolved as a mechanism to combat invading dsRNA viruses or to inhibit the activity of retrotransposons. Moreover, there is at least one gene in Drosophila, Stellate, that is regulated by dsRNA-mediated gene silencing in the testis (33). This chapter describes the application of RNAi to cultured Drosophila cells, with a particular emphasis on the imaging of the cytoskeleton and chromosomes in affected cells. Materials and methods are provided to enable the researcher to implement the design and production of dsRNA from polymerase chain reaction (PCR) templates, the culture of Drosophila cells and their treatment by RNAi, the analysis of target protein depletion by Western blotting, and the fixation and treatment of cells for microscopic imaging. The depletion of centrosomin (Cnn) a centrosomal protein that is required for mitotic centrosome assembly and function (34-37) from S2 cells is presented for example, but the technique is widely applicable to different targets and cell lines (7,17,38). Importantly, Drosophila cells in culture readily take up exogenous dsRNA, and there is no need to use carriers or transfection methods like those required with mammalian cell culture (7). Thus, RNAi holds great promise for the analysis of protein function in living cells.

Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases.

To perform preimplantation genetic diagnosis for women carrying heteroplasmic mitochondrial DNA (mtDNA) mutations, it is necessary to ensure that the proportion of mutant mtDNA diagnosed in the biopsied cell gives an accurate indication of the mutant load in the remaining embryo. A heteroplasmic mouse model, carrying NZB and BALB mtDNA genotypes, was used to study the relative proportions of each mtDNA genotype in the ooplasm and first polar body of mature oocytes, and between blastomeres of early cleavage stage embryos. The levels of heteroplasmy varied widely in the gametes compared with the maternal genotype. However, the distribution of the two mtDNA genotypes was virtually identical between the ooplasm and polar body of a mature oocyte, and also between the blastomeres of each 2-, 4- and 6-8-cell embryo. Therefore, the level of heteroplasmy diagnosed from the polar body of an unfertilized oocyte or from a single blastomere of an embryo is representative of the level in the embryo as a whole. Reliable results were obtained from both polar bodies and blastomeres, but the efficiency of diagnosis was greater with blastomeres. We conclude that preimplantation genetic diagnosis is feasible for mtDNA diseases, although it should be approached with caution, as it is possible that transmission of some pathogenic mutations could behave in a different manner.

Role of endogenous reverse transcriptase in murine early embryo development.

We report that a reverse transcriptase (RT) activity is present in early cleavage stage embryos as determined by a Polymerase chain reaction (PCR)-based detection assay. In an attempt to establish whether this activity plays a role in early embryonic development, we have blocked the endogenous RT by two independent approaches: (1) embryos were exposed to nevirapine, a highly specific nonnucleoside inhibitor of RT activity; (2) anti-RT antibody was microinjected into the nucleus of one blastomere of 2-cell embryos. When embryos were exposed to nevirapine in the developmental window between late 1-cell and 4-cell stages, development was arrested before the blastocyst stage. In contrast, development was not affected when embryos were exposed to nevirapine after the eight-cell stage. Developmental arrest was also induced when anti-RT antibody was microinjected in one blastomere of 2-cell embryos. Analysis of gene expression by RT-PCR in nevirapine-arrested 2-cell embryos revealed an extensive and specific reprogramming of gene expression, involving both developmentally regulated and constitutively expressed genes, compared to control embryos. These results support the conclusion that an endogenous RT activity is required in mouse early embryogenesis specifically between the late 1-cell and the 4-cell stage.

Do early cleavage-stage embryos demonstrate a culture media preference?

Objective: To evaluate and compare the cleavage rate and embryo quality of early cleavage-stage embryos from the same patient following culture in Preimplantation Stage-One Medium (P-1) and Quinn’s Cleavage Medium (QCM).

Incidence of chromosomal mosaicism in human embryos at different developmental stages analyzed by fluorescence in situ hybridization.

Chromosomal mosaicism has been reported in in vitro-cultured embryos at early cleavage stages, as well as in morulae and blastocysts. We have assessed the incidence and pattern of mosaicism during in vitro development of human embryos from early-cleavage stages to morula and blastocyst. Fifty spare embryos were fixed for fluorescence in situ hybridization (FISH) analysis for chromosomes X, Y, 13, 18, and 21 on days 2 or 3 (4- to 10-cell stage) (n = 16), on day 4 (morula stage) (n = 14), on day 5 (pre-expanded blastocyst) (n = 5), and the expanded blastocyst stages (n = 15). Blocked embryos (no cleavage observed within the last 24 hr) were not included. A total of 2367 cells were analyzed. Four early-cleavage stage embryos were found uniformly diploid; all of the others were mosaic for the chromosomes analyzed (mean diploid nuclei 48.3% +/- 28.7). All of the embryos at more advanced developmental stages, except one fully normal morula, had mosaic chromosome constitutions, with an increase in the percentage of diploid cells in morulae, pre-expanded, and expanded blastocysts, respectively (mean diploid nuclei 78.6% +/- 11.7, 66.0% +/- 20.8, 79.6% +/- 12.8), in comparison with earlier stages. Hypotheses about the origin of mosaicism and embryo regulation mechanisms will be discussed.

Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success

The use of cryoprotective agents for the conventional cryopreservation of human spermatozoa, oocytes, zygotes, early cleavage stage embryos and blastocysts is an integral part of almost every human IVF programme. Moreover, the cryopreservation of these types of cells by direct plunging into liquid nitrogen usually requires high cryoprotectant concentrations with consequent cytotoxic effects. This review covers the history of this problem, and in this light offers an explanation, through physico-chemical concepts, for one of the most recent developments in this area: the recovery of motile and potent spermatozoa after cryoprotectant-free vitrification.

Effects of Protein Source and Energy Substrates on the In Vitro Development of Bovine Embryos in a Two-step Culture System

In this study, we examined the effects of a two-step culture system, which involves the use of different culture media for early cleavage and later stage embryos, on the in vitro development of bovine embryos. We also investigated the effect of glucose, phosphate and citrate on the in vitro early developmental period of bovine embryos in a two-step culture system. Moreover, the supplementation of different protein sources (BSA-V, BSA-FAF and FBS) during IVC did not affect the frequency of blastocyst development. Using two-step culture, embryos were cultured in protein-free media for an initial 5 days. This was then followed by the same culture media or an FBS supplemented media. The developmental rates of blastocysts in the FBS containing group were significantly higher than in the replaced with no serum containing group. Embryos cultured in mSOF supplemented with 1.5 mM glucose plus 1.2 mM phosphate were significantly inhibited. The inhibition of developmental competence by glucose plus phosphate was consistent with the existence of 0.5 mM sodium citrate. This study indicates that a two-step culture system, which applies different conditions for early cleavage embryos, i.e., serum-free media, vs. later stage embryos, with serum containing media, may be effective for in vitro production systems. In addition, the developmental competence of bovine embryos was depressed in the presence of glucose plus phosphate as compared to either alone or the absence of both. Therefore, the avoidance of this negative effect should allow more optimal conditions to be developed for in vitro production.

Alkaline phosphatase activity in bovine oocytes and preimplantation embryos as affected by removal of the zona pellucida and culture medium constituents.

The aims of the present study were: (1) to characterize alkaline phosphatase (AP) activity in bovine oocytes and embryos with intact or removed zona pellucida (ZP); and (2) to evaluate the effect of culture medium constituents on AP activity. Alkaline phosphatase activity in non-matured and matured oocytes was most evident nearest the plasma membrane and perivitelline space. In more than 90% of two- to 16-cell embryos, AP activity was observed in the perivitelline space and at blastomere contacts. In blastocysts, AP activity was localized to the trophectoderm. Only after immunodissection was AP activity detected in the inner cell mass. Removal of the ZP by pronase or mechanical means reduced AP activity. Alkaline phosphatase activity was detected in evacuated ZP after mechanical removal. Specific constituents comprising the embryo culture medium altered AP activity. Alkaline phosphatase activity was reduced in eight- to 16-cell embryos and evacuated ZP cultured in CR1aa + 0.4% bovine serum albumin compared with embryos cultured in CR1aa alone or embryos co-cultured on a monolayer of Buffalo rat liver cells in the presence of 10% fetal bovine serum. The presence of AP activity at blastomere contacts and in evacuated ZP limits its usefulness as a marker for the differentiation of embryonic cells comprising the early cleavage-stage embryo.

Cadherins expression during gamete maturation and fertilization in the rat.

A role for adhesion molecules in gamete fusion, preceding fertilization, has been previously suggested. We investigated the presence of cadherins, Ca(2+) dependent cell-cell adhesion molecules, in rat oocytes and spermatozoa using an anti-pan-cadherin antibody and specific antibodies against the 3 classical cadherins: E- (epithelial), P- (placental), and N- (neural) cadherins. Electrophoretic separation was performed on samples of lysed oocytes of different stages: germinal vesicle oocytes, metaphase II eggs, newly fertilized and 2-cell embryos, as well as spermatozoa from testes, caput and cauda epididymis and ejaculate. Localization of cadherins was determined on intact, gametes by immunocytochemistry, using confocal microscopy. Immunoblotting with the pan-cadherin antibody revealed a major band of approximately 120 kD in all oocyte and sperm extracts. Oocytes presented E-cadherin at appropriate molecular weight but N-cadherin only as a specific 40 kD band. In sperm lysate, at all stages, both E- and N-cadherin were demonstrated as major protein bands but a series of lower molecular weight proteins (that may represent protein degradation) were also detected. Immunohistochemical evaluation showed that E- and N-cadherins are already present on the plasma membrane of immature unfertilized oocytes, although their concentration increases after fertilization in early cleavage stage embryos. Cadherin localization on spermatozoa changed during maturation from a dispersed pattern over the entire head plasma membrane of testicular spermatozoa to a restricted equatorial and post-acrosomal plasma membrane staining in ejaculated spermatozoa. These findings suggest a specific cadherin organization at the fusogenic domains of both gametes.

Efficient method for expressing transgenes in nonhuman primate embryos using a stable episomal vector.

Transgenesis in the nonhuman primate can enhance the study of human biology by providing animal models for the study of primate-specific physiology, pathophysiology, and embryonic development. Progress with this technology has been hindered by the inherent inefficiency of transgenesis, transgene silencing, and practical restrictions on the production of sufficient pronuclear stage nonhuman primate zygotes. We have developed a novel technique using an Epstein Barr virus (EBV)-based episomal vector to produce rhesus monkey (Macaca mulatta) embryos expressing a transgene. Plasmid DNA containing the latent origin of replication, oriP, and Epstein Barr Nuclear Antigen-1 (EBNA-1) of EBV, as well as a CMV IE-enhanced green fluorescent protein (eGFP) expression cassette, was introduced into rhesus embryos by direct pronuclear microinjection. We detected eGFP in early cleavage stage embryos (4-8 cell) and throughout the duration of culture (day 8-9 blastocysts) by epifluorescent microscopy. A 50% transduction rate was obtained with the EBV-based vector. Microinjected embryos expressed eGFP and retained their developmental capacity as evidenced by development to the blastocyst stage. EBV-based vectors present a novel and efficient means of delivering transgenes for the study of the molecular control of primate embryonic development.

Application of the zona-free manipulation technique to porcine somatic nuclear transfer.

The recent demonstration of a successful zona-free manipulation technique for bovine somatic nuclear transfer (NT) that is both simpler and less labor intensive is of considerable benefit to advance the applications of this technology. Here, we describe that this method is also applicable to porcine somatic NT. Porcine cumulus oocyte complexes were matured in TCM-199 medium before sequential removal of the cumulus and zonae. Zona-free oocytes were bisected using a microknife, and the halves containing the metaphase plate (as determined by Hoechst 33342 staining) were discarded. Each half cytoplast was agglutinated to a single granulosa cell (primary cultures grown in 0.5% serum for 2-5 days prior to use) in phytohaemagglutinin-P. Subsequently, each half cytoplast-granulosa cell couplet was simultaneously electrofused together and to another half cytoplast. Reconstructed embryos were activated in calcium ionophore A23187 followed by DMAP and were then individually cultured in microwells in NCSU-23 medium. On day 7 after activation, blastocyst yield and total cell numbers were counted. Of 279 attempted reconstructed NT embryos, 85.0 +/- 2.8% (mean +/- SEM; n = 5 replicates) successfully fused and survived activation. The blastocyst rate (per successfully fused and surviving embryo) was 4.8 +/- 2.3% (11/236; range, 0-12.8%). Total blastocyst cell count was 36.0 +/- 4.5 (range, 18-58 cells). The blastocyst rate and total cell numbers of parthenogenetically activated and zona-free control oocytes propagated under the same conditions was 11.6 +/- 3.9% (35/335 embryos; n = 3 replicates) and 36.8 +/- 5.2, respectively. Developmentally halted embryos that could still be evaluated on day 7 possessed 54.4 +/- 2.3% (53/96 embryos; n = 3 replicates) anucleate blastomeres, the latter representing 53.5 +/- 6.6% of the blastomeres in such embryos. In conclusion, blastocyst yield was independent of activation efficiency and was likely reduced by insufficient nuclear remodeling, reprogramming, imprinting, or other effects. The data also suggest that fragmentation was a considerable problem that could conceivably contribute to halted development in a high proportion of embryos. The results indicate that the zona-free manipulation technique can be successfully applied to pig somatic NT. Although such zona-free early cleavage stage embryos cannot be transferred to recipients at present, this technique permits simplification of the NT technique for application in basic research, until pig nonsurgical blastocyst transfer becomes a realistic option.

Sex ratio and birth weights of infants born as a result of blastocyst transfers compared with early cleavage stage embryo transfers

Objective: To analyze the birth weights and sex ratio of infants born as a result of blastocyst transfer and compare them with data resulting from the transfer of early-cleavage stage embryos.Design: Retrospective analysis.Setting: Monash IVF (private in vitro fertilization clinic).Patient(s): One hundred twenty-five infertile patients who became pregnant after IVF procedures involving blastocyst transfer.Intervention(s): None.Main Outcome Measure(s): Sex ratio and birth weights of infants born after blastocyst transfer.Result(s): The sex ratio of 129.6 for infants born after blastocyst transfer was not significantly different from the sex ratio calculated from data compiled by NPSU for births resulting from early cleavage stage embryo transfers at Monash IVF (100.6) and all other assisted conception units in Australia and New Zealand (97.9). No differences were observed in the combined mean birth weight of male and female infants born as a result of blastocyst transfers and early-cleavage stage embryo transfers.Conclusion(s): There is no evidence of abnormal fetal growth or a shift in the sex ratio for infants born as a result of blastocyst transfer when compared with the case of births resulting from early cleavage stage embryo transfers within our unit or all other assisted conception units in Australia and New Zealand.

Early cleavage versus blastocyst stage embryo transfer: is there any advantage?

Objective: Recently, blastocyst stage transfer has been suggested as a means of increasing implantation and pregnancy rates, and reducing multiple pregnancy rates in good prognosis patients undergoing IVF. Our study aims to evaluate the value of blastocyst stage transfer in a general, mixed, IVF population.

Use of embryo transfer and IVF to bypass effects of heat stress

Although heat stress has multiple effects to lower pregnancy rate in lactating dairy cows, a major pathway is in its effects on the early cleavage stage embryo. Conceptually, and in practice, higher pregnancy rates can be obtained with transfer of late cleavage stage embryos. The literature is reviewed, and conclusion is made that application of these technologies may be in part, a solution to this long-standing problem.

The vegetally localized mRNA fatvg is associated with the germ plasm in the early embryo and is later expressed in the fat body.

Vegetally localized RNAs in Xenopus oocytes have been implicated in the establishment of the primary germ layers and the formation and development of the primordial germ cells. fatvg mRNA is localized through the late pathway to the vegetal cortex. Like Vg1 mRNA fatvg is distributed throughout the entire cortex; however, unlike Vg1 there is a small fraction of the fatvg mRNA that is associated with the mitochondrial cloud. In early cleavage stage embryos, fatvg mRNA is associated with the germ plasm located at the tips of the vegetal blastomeres of the embryo. While several localized RNAs that follow the Message Transport Organizer (METRO) pathway have been found in the germ plasm in embryos, fatvg is a late pathway RNA that is associated with the germ plasm. In tadpoles, fatvg mRNA shows a novel pattern of expression which is distinct from the germ cell lineage and is detected at the dorso-anterior margin of the endodermal mass along the midline in two clusters of cells. fatvg mRNA expression is also detected later in the developing fat bodies, the major adipose tissues of the frog.

Growth and viability of human blastocysts in vitro

The transfer of a blastocyst established the first human clinical pregnancy following in vitro fertilization (IVF). Nine years later Cohen et al. reported pregnancies resulting from the transfer of cryopreserved human blastocysts. However, it was another six years before the first report of births resulting from the transfer of human blastocysts produced in vitro appeared in the medical literature. In the intervening period clinics have opted to transfer embryos at the early cleavage stage to the uterus, despite the fact that in vivo the embryo does not enter the uterus until two to three days later at the morula to blastocyst stage of development. The viability and potential for implantation of blastocysts is high, as indicated by the finding that more than 60% of in-vivo-derived blastocysts, recovered by uterine lavage following artificial insemination of fertile donors, implant and develop into viable fetuses when transferred to recipients. This is in stark contrast to the 10–20% of in-vitro-produced embryos transferred at the early cleavage stage of development that result in a live-birth. This reduction in viability following transfer of in-vitro-derived early cleavage stage embryos may have several possible explanations: (1) a failure of implantation due to poor synchronization between the embryo and the uterine endometrium; (2) a hostile environment in the uterus for early cleavage stage embryos; (3) sub-optimal in vitro culture conditions which result in a reduction in embryo viability; (4) the assumption that all oocytes retrieved in an IVF cycle have an equal ability to develop into viable embryos; and (5) the failure to identify the most viable embryo in a cohort. Certainly, improving culture conditions and laboratory techniques for developing high quality blastocysts routinely in vitro will not only address many of the above questions but will also improve the quality and viability of earlier stages of embryo development.

Type I Inositol 1,4,5-Trisphosphate (InsP 3) Sensitive Receptors are Prominently Expressed in the Blastomere Nuclei in Developing Human Preimplantation Embryo Beyond the 6–8 Cell Stage

Objective: Spontaneous rises in the intracellular Ca2+ occur in human preimplantation embryos and may regulate their development. The secondary messenger InsP3 and its receptor/s (InsP3R) are likely to play a major role in operating this process. These receptors are expressed in early cleavage stage embryos at peculiar sites and are detected on Western blots as a 260 KD protein (Mol Hum Reprod 1999;5:441–451). The aim of this study was to investigate and compare distribution of type I InsP3 in human cleavage stage embryos derived from in vivo and in vitro matured (IVM) oocytes using confocal laser scanning microscopy (CLSM).