Blastomere Cells Research Articles

146 EFFECT OF MATERNAL AGE AND PARITY ON THE PRESENCE AND DISTRIBUTION OF CADHERIN-E IN HAMSTER EMBRYOS DURING PRE-IMPLANTATION DEVELOPMENT AND TRANSPORT

Our knowledge of the factors that contribute to in vivo embryo development and transport are still incomplete. Although there is solid evidence that the hormonal state of the female regulates ovum transport in mammals, recently, because of significant differences between the rate of oviduct transport of fertilized and unfertilized eggs in several mammalian species, there have been some reports showing that the conceptus itself may auto regulates its own transport. During the initial phases of embryonic development the distribution of E-cadherin, a calcium dependent cell adhesion molecule, changes from a general distribution over the whole surface of the blastomeres to become almost exclusively localized over the points of contact between blastomeres. This change on E-cadherin localization has been related to the beginning of embryo compactation and is considered as the initial stage of differentiation. We have recently shown that pre-implantation embryo development and transport is highly synchronous in nuliparous young females (NYF), but not in adults. Nuliparous adult (8 months) females (NAF) and multiparous adult females (MAF) show significant asynchrony and retard on early embryo development. In NYF, all 8 cells embryos reached the uterus by 62 h after coitus. In adult females, both nuliparous and multiparous, a considerable number of 8 cells embryos fail to migrate into the uterus even at 67 h after coitus. E-cadherin distribution was studied by indirect immunofluorescence and confocal microscopy during preimplantation embryo development in three groups, NYF, MAF, and NAF, of regularly cycling golden hamster (Mesocricetus auratus) females. Females were mated with males of proven fertility. Only 15 minutes were allowing for mating. Time of ejaculation was registered. Females were sacrificed from 60 to 69 h after coitus. Corpora lutea were counted in both ovarian surfaces. Oviducts and uterine horns were flushed separately and embryo number, stage of development, and distribution were recorded. Cell numbers, cadherin distribution and the number of cadherin-dependent cell to cell contacts were established in at least 20 embryos per group and at each of the different hours of the study. Presence and distribution of E-cadherin on 4-, 6-, and 8-cell blastomeres embryos differs accordingly with the parity and age of the females. Differences are particularly important at the eight-cell stage 80 to 93% of the embryos from NYF showed normal cadherin dependent cell contacts with all neighboring cells. In the case of multiparous adult females (MAF) only 52 to 68% of the embryos showed normal cell to cell contacts. These results will be discussed in relation to the respective role that the female hamster and the conceptus itself play in early embryo development and transport. Our results also indicate that in spite of the significantly higher ovulation rate of old females, their reproductive efficiency were significantly lower than that of NYF.

214 DIFFERENT GENE EXPRESSION OF INDIVIDUAL BLASTOMERES IN EARLY MOUSE EMBRYO DETECTED BY REAL TIME PCR

The objective of this study was to detect gene expression differences among blastomeres from 4- and 8-cell stage mouse embryos by a sensitive method. In earlier studies gene expression was determined in pooled or single embryos. With the higher levels of precision afforded by our method, we could quantify mRNA in single blastomeres. For comparison of gene expression, different inner cell mass specific (Oct-4, Nanog) and trophectoderm-specific (Glut-3) genes and the housekeeping beta-actin gene (for reference) were chosen. Late 4- and early 8-cell stage embryos were obtained from oviducts 58 and 66 h post-hCG, respectively. The embryos were collected in CZB-HEPES and the zonae pellucidae were dissolved by acid Tyrode solution. Blastomeres were separated mechanically from each other by gentle pipetting with a fire-polished glass capillary. Messenger RNA was isolated from the frozen blastomeres using the Dynabeads® mRNA DIRECTTM Micro Kit (Kvalitex Kft., 1136 Budapest, Hungary) following the manufacturer's protocol. The reverse transcription reactions were performed at 25°C for 10 min, and then at 42°C for 1 h followed by 99°C for 5 min. Real-time PCR was performed in an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, 1094 Budapest, Hungary). Reaction mixtures, which were separated for each gene, consisted of QuantiTect iQ SYBR Green Supermix (Bio-Rad Laboratories), 500 nm of both primers, and a 1/8 aliquot of the blastomere cDNA. The cDNA template was denatured by heating to 95°C for 2 min and amplified by 50 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s, with a single fluorescence measurement at each cycle. When the reaction was finished, melting curves were plotted to confirm product purity. By this procedure we could measure the expression level of four genes in six 4- and 8-cell-stage embryos. Glut-3 expression was not detectable in the blastomeres of 4-cell stage embryos. In spite of the 1.3–1.5-fold technical error, Oct-4 and Nanog showed 10-fold and 9-fold differences, respectively. Among the blastomeres of the same embryo, one of the 4-cell-stage showed notably high expression of Oct-4. In the 8-cell stage blastomeres Oct-4 expression showed slight differences (2–8-fold), and Nanog and Glut-3 showed 28-fold and 12-fold differences, respectively. Two of the 8-cell blastomeres showed notably high expression of Nanog. In conclusion, this method was accurate and sensitive enough to measure the expression of different genes in a single blastomere. We found that the mRNA levels of Oct-4 and Nanog have remarkably discrete differences within the blastomeres of a 4-cell-stage embryo. Expression of Nanog gene showed notable differences within blastomeres of an 8-cell-stage embryo. These differences may show a very early sign of the cell fate determination. This work was funded by the Wellcome Trust International Senior Fellowship and Bio-Rad Laboratories.

Impact of the presence of one or more multinucleated blastomeres on the developmental potential of the embryo to the blastocyst stage

A retrospective study of 5,982 embryos in 619 blastocyst-stage embryo transfer cycles revealed that detection of multinucleated blastomeres either on day 2 or 3 signifies a poor prognosis for blastocyst formation and that no good-quality blastocyst can be expected from an embryo with more than one multinucleated blastomere. Patients should be counseled regarding a poor prognosis when multiple embryos with multinucleated blastomeres are present.

Role of a spatial distribution of IP3 receptors in the Ca2+ dynamics of the Xenopus embryo at the mid‐blastula transition stage

Periodic calcium activity correlates temporally with the onset of gene expression in the embryo, suggesting a causal relation between these two events. Calcium transients are elicited by the action of fibroblast growth factor (FGF) through the activation of phospholipase C. In this work, we present a reaction-diffusion model that extends our previous results on the generation of calcium oscillations for a single and two coupled blastomere cells to a meridian of the Xenopus embryo at the mid-blastula transition. In the model, all cells are subject to the same amount of FGF and contain the same concentration of intracellular components, except for the amount of IP(3) receptors (IP3R). A bell-shaped distribution of IP3R produces the correct shape of the calcium transients experimentally observed in the Xenopus blastula at stage 8 (mid-blastula transition stage). The model is also capable of predicting period and amplitude values close to the experimental values. In our model, calcium transients induce spatially localized ERK periodic transients that could activate specific nuclear genes, allowing for the regional differentiation of the cells in the zone under the influence of the calcium signal.

Spatial arrangement of individual 4-cell stage blastomeres and the order in which they are generated correlate with blastocyst pattern in the mouse embryo

In the unperturbed development of the mouse embryo one of the 2-cell blastomeres tends to contribute its progeny predominantly to the embryonic and the other to the abembryonic part of the blastocyst. However, a significant minority of embryos (20–30%) do not show this correlation. In this study, we have used non-invasive lineage tracing to determine whether development of blastocyst pattern shows any correlation with the orientation and order of the second cleavage divisions that result in specific positioning of blastomeres at the 4-cell stage. Although the orientation and order of the second cleavages are not predetermined, in the great majority (80%) of embryos the spatial arrangement of 4-cell blastomeres is consistent with one of the second cleavages occurring meridionally and the other equatorially or obliquely with respect to the polar body. In such cleaving embryos, one of the 2-cell stage blastomeres tends to contribute to embryonic while the other contributes predominantly to abembryonic part of the blastocyst. Thus, in these embryos the outcome of the first cleavage tends to correlate with the orientation of the blastocyst embryonic–abembryonic axis. However, the order of blastomere divisions predicts a specific polarity for this axis only when the earlier 2-cell blastomere to divide does so meridionally. In contrast to the above two groups, in those embryos in which both second cleavage divisions occur in a similar orientation, either meridionally or equatorially, we do not observe any tendency for the 2-cell blastomeres to contribute to specific blastocyst parts. We find that all these groups of embryos develop to term with similar success, with the exception of those in which both second cleavage divisions occur equatorially whose development can be compromised. We conclude that the orientations and order of the second cleavages are not predetermined; they correlate with the development of blastocyst patterning; and that the majority, but not all, of these cleavage patterns allow equally successful development.

Effect of cryopreservation on mitochondrial DNA of zebrafish ( Danio rerio) blastomere cells

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programmes for endangered species. However, despite the growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells very often remains poor. Many studies have been devoted to the mechanisms of cryodamage. It is known that cryopreservation causes extensive damage to membranes; reduce the metabolic activity of cells; and disturbs the mitochondrial bioenergetical processes of cells. But few investigations on the genetic stability of cells during cryopreservation have been performed, and the role of any genetic impact cryopreservation needs to be determined. Some indirect data in the literature suggests that progress in this field might come from investigating freezing damage to mitochondrial DNA (mtDNA), nuclear DNA and other genome-related structures. In this study, zebrafish ( Danio rerio) blastomeres were treated in three different ways: control suspension of blastomere cells in phosphate buffered saline; equilibration of blastomeres with 2 M dimethyl sulfoxide (Me 2SO) for 1 h at room temperature and cryopreservation using Me 2SO as a cryoprotectant. Mitochondrial DNA was analysed in fresh cells and after the different treatments. Two different loci of mtDNA were amplified with the help of PCR and sequenced. The sequences were analysed and nuclear base substitutions were counted for both control and treated samples. The results showed that cryopreservation significantly increased the frequency of mutations (0.78 ± 0.27% in comparison to 0.16 ± 0.25% of control), whilst 2 M Me 2SO treatment did not bring a significant increase in frequency of mutations (0.24 ± 0.28%). The distributions of the mutation locations were analysed. More investigations are needed to determine whether optimisation of cryopreservation protocol is possible to reduce these adverse effects; whether such mutations interfere with overall function of the cells; whether similar changes also occur in the nuclear DNA and whether such mutations happen in other species. Meanwhile, it is important to be cautious in making judgements of the effect of cryopreservation technique in assisted reproduction. This is the first report on the effect of cryopreservation on mtDNA.

Transcript profiling during preimplantation mouse development

Studies using low-resolution methods to assess gene expression during preimplantation mouse development indicate that changes in gene expression either precede or occur concomitantly with the major morphological transitions, that is, conversion of the oocyte to totipotent 2-cell blastomeres, compaction, and blastocyst formation. Using microarrays, we characterized global changes in gene expression and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany and likely underlie these transitions. The analysis confirmed previously described processes or events, but more important, EASE revealed new insights. Response to DNA damage and DNA repair genes are overrepresented in the oocyte compared to 1-cell through blastocyst stages and may reflect the oocyte's response to selective pressures to insure genomic integrity; fertilization results in changes in the transcript profile in the 1-cell embryo that are far greater than previously recognized; and genome activation during 2-cell stage may not be as global and promiscuous as previously proposed, but rather far more selective, with genes involved in transcription and RNA processing being preferentially expressed. These results validate this hypothesis-generating approach by identifying genes involved in critical biological processes that can be the subject of a more traditional hypothesis-driven approach.

General Concepts About Stem Cells as Potential Therapeutic Agents

After completing this article, readers should be able to: 1. Define stem cells. 2. Describe the two classes of stem cells. 3. Delineate the method for isolating human embryonic stem cells. 4. Define therapeutic cloning. 5. Describe the potential role for embryonic stem cells in treating diabetes mellitus. Stem cells display the ability to self-renew (to divide and give rise to other stem cells) and to produce offspring that mature along distinct differentiation pathways to form cells that have specialized functions. In vertebrates, stem cells classically have been divided into two groups: embryonic stem (ES) cells and somatic or adult stem cells. ES cells are derived from cells in preimplantation blastocysts. Adult stem cells represent the self-renewing populations residing in many tissues and organs, including bone marrow, brain, retina, skin, intestine, liver, kidney, and pancreas. ES cells initially were isolated from cultured murine blastocysts, and the murine system continues to be the most amenable for ES cell line isolation. In the murine system, ES cells display the potential to repopulate (to some extent) all tissues of the embryo, including germ cells. Because adult stem cell populations potentially can be derived from these formed tissues and organs, adult stem cells by definition can be derived from ES cells. No evidence has yet been presented that adult stem cells can give rise to ES cells, although one population of adult stem cells—multipotent adult progenitor cells—display features strikingly similar to ES cells. The fertilized oocyte (zygote) is the “mother” of all stem cells. All the potential for forming all cells and tissues of the body, including the placenta and extraembryonic membranes, are derived from this cell. Thus, the zygote is a totipotent cell. The first few cleavage stage divisions produce blastomere cells that retain totipotent potential. However, by the 64-cell stage of mouse development, the cells begin to …

Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

In order to investigate the effects of different kinds of nuclear recipients from Kunming (KM) mouse on developmental potential of somatic nuclear transfer embryos, the enucleated M II oocytes, enucleated zygotes and 2-cell blastomere were used to produce cloned mouse embryos. Using fibroblast deriving from C57/BL6 ear tissue as nuclear donor, we produced cloned embryos by transferring the fibroblast nuclei into enucleated KM mouse oocytes (single nuclear transfer, SNT), transferring pronuclei from the SNT embryos into enucleated KM zygotes (nuclear into zygote, NZ), and 2-cell blastomere nuclei from SNT embryos into enucleated KM mouse oocytes (nuclear into oocytes, NO); tetraploid embryos (tetraploid embryos, TE) were obtained by fusing two blastomeres, one is from the SNT cloned embryos, and the other from normal 2-cell KM mouse embryos. In group SNT, the cloned embryos could not develop beyond 8-cell stage and the rate of 8-cell stage is only 0.3%; in group NO, the reconstructed embryos could develop to morula stage, the rate of 8-cell stage was significantly greater than that of SNT group (P < 0.05); in group NZ, the development rate was further improved, and the reconstructed embryos could develop into blastocyst stage, the rate of blastocyst was 1.9%; in group TE, as high as 62.3% of the reconstructed embryos could develop into blastocyst. Results suggested that different nuclear recipients could significantly affect the developmental potential of cloned mouse embryos; KM M II oocyte cytoplasm was not so effective as zygotes to reprogram the mouse somatic cell nuclei; serial nuclear transfer could improve the developmental potential of cloned mouse embryos.

Differential Influences in Sizes and Cell Cycle Stages of Donor Blastomeres on the Development of Cloned Rabbit Embryos

Experiments were conducted to evaluate the effect of blastomere diameters and cell cycle stages on the subsequent development of nuclear transplant rabbit embryos (NT-embryos) using nuclei derived from the 16- or 32-cell stage embryos. All blastomeres and NT-embryos were cultured individually in modified Ham's F-10 medium supplemented with 10% rabbit serum (RS) at 38°C and 5% CO 2 in air. The diameter of blastomeres from 16-cell stage embryos was found twice of those from 32-cell stage (51 vs 27 μm). Significant differences were observed in cleavage rates (>3 divisions) in the isolated single blastomeres (54 vs 48 for 16-cell; 28 vs 14 for 32-cell, p 3 divisions) of the NT-embryos were greater in the small nuclear donors than in the large donors (73 vs 55%, p 0.05). In conclusion, it appeared that no difference in the developmental competence between large and small isolated blastomeres was observed. When smaller 16-cell stage blastomeres were used as nuclear donor, the cleavage rate or development of NT-embryos was improved and was compromised when 32-cell stage blastomeres were used. Therefore, control nuclear stage of the donor cell at G 1 phase in preactivated nuclear recipients seemed to be beneficial for the cleavage rate of the reconstructed embryo in the 16-cell transplant, but not for subsequent morula or blastocyst development.

Early patterning of the mouse embryo--contributions of sperm and egg.

The first cleavage of the fertilised mouse egg divides the zygote into two cells that have a tendency to follow distinguishable fates. One divides first and contributes its progeny predominantly to the embryonic part of the blastocyst, while the other, later dividing cell, contributes mainly to the abembryonic part. We have previously observed that both the plane of this first cleavage and the subsequent order of blastomere division tend to correlate with the position of the fertilisation cone that forms after sperm entry. But does sperm entry contribute to assigning the distinguishable fates to the first two blastomeres or is their fate an intrinsic property of the egg itself? To answer this question we examined the distribution of the progeny of early blastomeres in embryos never penetrated by sperm - parthenogenetic embryos. In contrast to fertilised eggs, we found there is no tendency for the first two parthenogenetic blastomeres to follow different fates. This outcome is independent of whether parthenogenetic eggs are haploid or diploid. Also unlike fertilised eggs, the first 2-cell blastomere to divide in parthenogenetic embryo does not necessarily contribute more cells to the blastocyst. However, even when descendants of the first dividing blastomere do predominate, they show no strong predisposition to occupy the embryonic part. Thus blastomere fate does not appear to be decided by differential cell division alone. Finally, when the cortical cytoplasm at the site of sperm entry is removed, the first cleavage plane no longer tends to divide the embryo into embryonic and abembryonic parts. Together these results indicate that in normal development fertilisation contributes to setting up embryonic patterning, alongside the role of the egg.

Experimental analysis of second cleavage in the mouse.

Mammalian conceptuses typically have an approximately regular tetrahedral shape at the 4-cell stage. In the rabbit, this has been attributed to both 2-cell blastomeres dividing meridionally, but with the animal-vegetal axis of the second blastomere to divide rotating through roughly 90 degrees before or during cytokinesis. The aim of the present study was to ascertain whether this was also true for the mouse. First, the distribution in regular tetrahedral 4-cell conceptuses of fluorescent microspheres applied to the vegetal polar region of one or both blastomeres at the 2-cell stage was analysed. Second, the ability of 2-cell stages to form regular tetrahedral 4-cell conceptuses after the previtelline space had been gelated to prevent blastomeres from rotating was also investigated. Neither experiment yielded evidence supporting blastomere rotation during second cleavage. Rather, the findings were consistent with the regular tetrahedral form of 4-cell conceptus resulting from meridional division of one blastomere and approximately equatorial division of the other. Second cleavage in the mouse typically yields 4-cell conceptuses with three distinct types of blastomere. While both products of the meridional division include all axial levels of the zygote, those of the equatorial division acquire only its vegetal or animal half.

Preimplantation Genetic Diagnosis

Genetics & Birth Defects| June 01 2002 Preimplantation Genetic Diagnosis AAP Grand Rounds (2002) 7 (6): 70–71. https://doi.org/10.1542/gr.7-6-70 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Preimplantation Genetic Diagnosis. AAP Grand Rounds June 2002; 7 (6): 70–71. https://doi.org/10.1542/gr.7-6-70 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search nav search search input Search input auto suggest search filter All PublicationsAll JournalsAAP Grand RoundsPediatricsHospital PediatricsPediatrics In ReviewNeoReviewsAAP NewsAll AAP Sites Search Advanced Search Topics: preimplantation diagnosis Source: Kanavakis E, Traeger-Synodinos J. Preimplantation genetic diagnosis in clinical practice. J Med Genet. 2002;39:6–11. Preimplantation genetic diagnosis (PGD) involves the testing of an in vitro fertilization (IVF) derived embryo at risk for a specific genetic disorder, thereby enabling selection of unaffected embryos for implantation. This review article from Aghia Sophia Children’s Hospital, Athens, Greece, points out that PGD is an alternative genetic test to chorionic villus sampling (CVS) or amniocentesis, genetic testing techniques used in an already established pregnancy that carries an increased risk for a genetic disorder. Embryonic cells for testing may be obtained from polar bodies of the oocyte/zygote stage, trophectoderm cells from blastocyst stage embryos, and blastomere cells from cleavage stage embryos. Although polar bodies are useful for detecting maternally derived numerical chromosome abnormalities associated with advancing maternal age, it is usually not preferred because the paternal contribution is unknown. Blastocyst biopsy has not yet been reported for PGD because only 40–50% of in vitro embryos successfully reach the blastocyst stage. The cleavage stage embryo has 6–8 blastomere cells on the 3rd day post-insemination, 1 cell of which is used for testing, and is the most commonly used source despite the limited amount of material available for analysis. Single cell diagnosis is the most difficult technical part of PGD and genetic diagnosis must be completed and embryo transfer accomplished within 48 hours so that embryo viability is not compromised. PGD applications include diagnosis of chromosome abnormalities using fluorescence in situ hybridization (FISH) and monogenic disorders using polymerase chain reaction (PCR). Approximately half of all PGD done to date have been done because of advanced maternal age and the associated increased risk for chromosome abnormalities. The monogenic disorders for which PGD has been done include disorders which are autosomal recessive (eg, hemoglobinopathies, Tay-Sachs disease, and cystic fibrosis), autosomal dominant (eg, myotonic dystrophy and Charcot-Marie-Tooth disease 1A), and X-linked (eg, muscular dystrophy, hemophilia, and fragile X syndrome). Although PGD has been valuable and worthwhile in averting serious inherited diseases for many couples, it will probably not replace first and second trimester prenatal diagnosis because it is a technically difficult, complex, labor intensive, and expensive procedure. PGD appears to be safe without adverse consequences on early development based on the experience of 250 babies born after PGD1,2; however, IVF is the only way in which a blastomere cell can be obtained for testing, even for those couples without fertility problems. PGD results in a low rate of successful pregnancies: the testing is successful in about 80–90% of blastomere cells obtained and only 50% have normal results. Consequently, approximately 40–50% of tested embryos are suitable for transfer/implantation with pregnancies resulting in no more than 33% of all cycles initiated for IVF.3,4 As the authors indicate, at this time PGD is most appropriate for couples who have a fertility problem and a significantly increased risk for having a child with a severe genetic disorder. PGD may be worthwhile for couples who have an affected child, have... You do not currently have access to this content.

Electrofusion of Blastomeres of Hamster 2-Cell Embryos and Dynamic Changes of the Cytoskeletal Distribution.

Cell fusion is an important process in current animal biotechnology. This study was designed to establish optimal parameters for the electrofusion of hamster 2-cell blastomeres and to examine the dynamic changes in the cytoskeletal distribution using fluorescence staining. Various electric fields (70-2000 V/mm) and durations (50-1000 μsec) of electric pulse were applied. Electric fields higher than 300 V/mm or of duration longer than 200 μsec caused disruption of the blastomere. Blastomere fusion occurred at 70-170 V/mm of 100 μsec duration, with high yields (91%). However, electrofused embryos did not develop. Fluorescence observations showed that the control 2-cell embryos possessed a dense network of microtubules around the nucleus and abundant microfilaments at the cell-to-cell contact region. In the fused embryos, two domains of cytoskeleton, consisting of both microfilaments and microtubules, concentrated around each nucleus were observed. In the embryos which failed to fuse, however, a thin layer of microtubules and very thick bundle of microfilaments appeared at the cell-to-cell contact region. The results suggest that blastomere fusion is accompanied with cytoskeletal reorganization and that dynamic changes of the cytoskeleton occur in different modes between fused and non-fused embryos.

Ploidy of Bovine Nuclear Transfer Blastocysts Reconstructed Using In Vitro Produced Blastomere Donors

The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro development. Because little is known about ploidy errors in nuclear transfer embryos, this was examined using embryos reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6-dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blastomeres from day 4-5 in vitro produced donors. The embryos were cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 +/- 20.2 (mean % +/- SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8%) were potentially abnormal because in these, 1.1%, 1.4%, 5.3%, 7.5%, 26.3%, 30.4%, and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors comprised mainly triploid (8.2 +/- 10.3 [0-26.3]: % +/- SD [range]) and tetraploid (10.6 +/- 19.9 [0-54.9]) nuclei with other ploidy combinations accounting for only 0.9 +/- 2.1 [0-2.1]% of deviant nuclei. The proportion of completely normal nuclear transfer embryos was no less than those produced by in vitro fertilization but the distribution of chromosome abnormalities was different (p = 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chromosome abnormalities to embryonic loss in the remaining embryos deserves further investigation.

Predictive value of 72-hour blastomere cell number on blastocyst development and success of subsequent transfer based on the degree of blastocyst development

Objective: To determine the predictive value of 72-hour blastomere cell number on blastocyst development and to compare success rates of subsequent transfer based on the degree of blastocyst development. Design: Retrospective clinical study. Setting: Private assisted reproductive technology center. Patient(s): Ninety-three women aged 32.0 ± 5.1 years undergoing oocyte retrieval for IVF. Intervention(s): Bipronucleate oocytes obtained from IVF were grown for up to 168 hours after fertilization and subsequently transferred at the blastocyst stage. Main Outcome Measure(s): Percentages of embryos developing to blastocyst from 72-hour embryos by blastomere cell number and subsequent implantation and pregnancy rates of transferred blastocysts. Result(s): Rates of blastocyst formation and expansion increased as cell numbers at 72 hours increased. Implantation rates were 43% for embryos transferred to women receiving only expanded blastocysts and 17% for embryos transferred to women receiving one or more less developed blastocysts. Pregnancy rates were higher for women receiving only expanded blastocysts than for women receiving one or more less developed blastocysts, although the difference was not significant. Conclusion(s): More developed 72-hour embryos are more likely to become blastocysts and expand. Implantation rates are greater for the transfer of expanded rather than unexpanded blastocysts.

Transition of the blastomere cell cycle from cell size-independent to size-dependent control at the midblastula stage in Xenopus laevis.

Dissociated animal cap blastomeres of Xenopus laevis blastulae were cultured at a low Ca level (1 microM) from 9th to 18th cell cycle at 22 +/- 1 degrees C and observed by a time-lapse video recorder. Blastomeres cleaved unequally to increase variability in cell size as cell cycles progressed, but synchronously at a constant cell cycle time of about 30 min up to the 12th cleavage in diploid cells, and up to the 13th cleavage in haploid cells, regardless of their cell sizes. Thereafter, blastomeres cleaved asynchronously at varying cell cycle times in proportion to the inverse square of their radii. The transition from the cell size-independent to -dependent cell cycles occurred at the critical cell radius, 37.5 microm for the diploid and 27.9 microm for the haploid. While the protein synthesis inhibitor, cycloheximide (CHX) lengthened cell cycle times two- to six-fold, epidermal growth factor (EGF) had no significant effect on the cell cycle. CHX-treated blastomeres synchronously cleaved at a constant cell cycle time of 60 min up to the 12th cleavage. Thereafter, cell cycle times became variable in proportion to the inverse square of radii in the presence of CHX at 0.10-0.14 microg/ml, but to the inverse cube of radii at 0.18 microg/ml. The critical cell size of CHX-treated blastomeres for the transition from cell size-independent to -dependent cell cycles remained the same as that of untreated blastomeres. Frequency distributions of cell cycle times of synchronous cell cycles were monomodal with the peak at 30 min, except for CHX-treated blastomeres with the peak at 60 min. In contrast, frequency distributions of asynchronous cell cycles were polymodal with peaks at multiples of a unit time of 30-35 min. To explain these results, we propose that blastomere cytoplasm has 30-min cycles that repeatedly produce mitosis promoting factor (MPF) in a quantity proportional to the cell surface area. MPF is neutralized when it titrates a nuclear inhibitor present in a quantity proportional to the genome size, and sequestered in the nucleus. When the total amount of MPF produced exceeds the threshold required to titrate all of the inhibitor, mitosis is initiated.

Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8-cell (demi-embryo) parthenogenetic and four 8-cell stage (demi-embryo) IVF-derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8-cell stage) and a whole IVF-derived embryo (8-cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF-derived blastomeres with parthenogenetic blsatomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVF-derived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8-cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8-cell parthenogenetic and two 4-cell IVF-derived blastomeres or eight 16-cell parthenogenetic and four 8-cell IVF-derived blastomeres were constructed. Parthenogenetic demi-embryos were aggregated with sexed (male) IVF demi-embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona-free) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4-cell and four 8-cell chimeric embryos compared with 10% for intact 8-cell parthenogenetic embryos and 15% for intact 16-cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona-free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypically- chimeric viable male calf was born. We conclude that the IVF-derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentally competent. Mol. Reprod. Dev. 53:159–170, 1999. © 1999 Wiley-Liss, Inc.

Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos.

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8-cell (demi-embryo) parthenogenetic and four 8-cell stage (demi-embryo) IVF-derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8-cell stage) and a whole IVF-derived embryo (8-cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF-derived blastomeres with parthenogenetic blastomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVF-derived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8-cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8-cell parthenogenetic and two 4-cell IVF-derived blastomeres or eight 16-cell parthenogenetic and four 8-cell IVF-derived blastomeres were constructed. Parthenogenetic demi-embryos were aggregated with sexed (male) IVF demi-embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona-free) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4-cell and four 8-cell chimeric embryos compared with 10% for intact 8-cell parthenogenetic embryos and 15% for intact 16-cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona-free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypically-chimeric viable male calf was born. We conclude that the IVF-derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentall

Multiple genetic diagnoses from single cells using multiplex PCR: reliability and allele dropout.

We used a multiplex fluorescent PCR system containing seven primer sets on single cells from three different cell types (buccal, corneal and blastomere cells) and more than 3500 heterozygous alleles to investigate reliability and extent of allele dropout in multiplex PCRs at the single cell level. All three cell types gave similarly high reliability, accuracy and allele dropout rates, with similar reliability between singleplex and multiplex PCRs. Allele dropout was also consistent between the three cell types and did not significantly increase as allele size increased. These results indicate that multiplex fluorescent PCR is a reliable and accurate method of obtaining multiple diagnosis (eight chromosomes simultaneously) from single cells and maximizes the information available from single cell analysis.