Water and DMSO membrane permeability characteristics of in-vivo- and in- vitro-derived and cultured murine oocytes and embryos

Although embryo cryopreservation is routine for many mammalian species, it is important to know how the fundamental cryobiology of these cells changes with development. Progressive cleavage divisions result in a reduction in the blastomere surface area available for water and cryoprotectant mass transport. Therefore, the membrane permeability of murine oocytes, zygotes, 2-cell, 4-cell, and 8-cell embryos to water (Lp), and dimethylsulphoxide (PDMSO), and the reflection coefficient, sigma (sigma) were determined. Oocytes or zygotes were recovered, cumulus cells removed, then cultured until use. Oocytes and embryos were immobilized and perfused with treatment solutions at 24 degrees C. Osmotically induced cell volume changes over time were videotaped followed by image analysis. The Lp values in the presence of dimethylsulphoxide (DMSO) were 0.77, 0.81, 0.94, 0.86, and 1.10 microm/min/atm, and the PDMSO values were 1.85, 2.04, 2.41, 1.95, and 1.25x10(-3) cm/min for oocytes, zygotes, 2, 4, and 8-cell embryos respectively. The Lp values in the presence of DMSO were significantly (P < 0.05) higher than those in the absence of DMSO. Treating the whole embryo as a single osmotic entity leads to significantly (P < 0.05) elevated PDMSO estimates relative to those based upon measurements of individual blastomeres. These data indicate that both Lp and PDMSO estimates are lower when predicted on an individual blastomere basis. The data also show that neither Lp nor PDMSO differ among oocytes, zygotes, 2-cell and 4-cell embryos. However, the significantly higher Lp and lower PDMSO of the 8-cell stage support the hypothesis that fundamental cryobiological differences may require developmental stage- specific embryo cryopreservation protocols.      

Computer-controlled, multilevel, morphometric analysis of blastomere size as biomarker of fragmentation and multinuclearity in human embryos

BACKGROUND: Little is known about blastomere size at different cleavage stages and its correlation with embryo quality in human embryos. Using a computer system for multilevel embryo morphology analysis we have analysed blastomeres of human embryos and correlated mean blastomere size with embryonic fragmentation and multinuclearity. METHODS: A consecutive cohort of 232 human 2-, 3- and 4-cell embryos from patients referred for ICSI treatment were included. Sequences of digital images were taken by focusing at 5- micro m intervals through the embryo. Blastomere sizes and number of nuclear structures were evaluated based on these sequences. The degree of embryonic fragmentation was evaluated by normal morphological assessment prior to transfer and correlated to the blastomere sizes. RESULTS: As a result of normal cell cleavage, mean blastomere size decreased significantly from a volume of 0.28 x 10(6) microm(3) at the 2-cell stage to 0.15 x 10(6) microm(3) at the 4-cell stage (P < 0.001). Mean blastomere size decreased significantly (P < 0.001) with increasing degree of embryonic fragmentation, where highly fragmented embryos showed a 43-67% reduction in blastomere volume compared with embryos with no fragmentation. Multinucleated blastomeres were significantly larger than non-multinucleated blastomeres (P < 0.001). On average, multinucleated blastomeres were 51.5, 67.8 and 73.1% larger than their non-multinucleated sibling blastomeres at the 2-, 3- and 4-cell stage, respectively. Furthermore, the average volume of non-multinucleated blastomeres originating from multinucleated embryos was significantly smaller than the average volume of the blastomeres from mononucleated embryos (P < 0.001). CONCLUSIONS: The results of this study show that the average blastomere size is significantly affected by degree of fragmentation and multinuclearity, and that computer-assisted, multilevel analysis of blastomere size may function as a biomarker for embryo quality.     

Experimental analysis of second cleavage in the mouse

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.     

Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres

Summary A detailed fate map of all the progeny derived from each of the blastomeres of the 4- and 8-cell stage South African clawed frog (Xenopus laevis) embryo is presented. Each “identified” blastomere that results from stereotypic cleavages has a characteristic set of progeny that distinguishes it from the other blastomeres of the embryo. The 4-cell dorsal (D) blastomere is the major progenitor of the stomodeum, cement gland, retina, notochord, head somite, pharynx and liver. The 4-cell ventral (V) blastomere is the major progenitor of the trunk and fin epidermis, ventral somite, nephrotome, lateral plate mesoderm and proctodeum. The other organs are derived from both blastomeres. At the next cell division, the animal hemisphere daughters of both blastomeres (D1 and V1, respectively) become the major progenitors for head ectodermal and mesodermal structures, and the vegetal hemisphere daughters become the major progenitors for trunk mesodermal (D2) or trunk endodermal (V2) structures. Semiquantitative lineage diagrams, using data from this and from previous studies demonstrate that as cleavage proceeds from the 2- to the 32-cell stage, the progenitors for particular organs or for specific regions of organs segregate into defined regions of the blastula. To determine whether this segregation is related to the position of the blastomere or to its geneological lineage, we compared the fates of radial 8-cell blastomeres to those of stereotypic 8-cell blastomeres. Radial blastomeres have fates nearly equivalent to the sum of the two 16-cell blastomeres that occupy the same position in the embryo, demonstrating that fate depends upon blastomere position rather than lineage.     

The frequency and developmental capability of human embryos containing multinucleated blastomeres

The frequency of multinucleated blastomeres (MNB) in 2- and 4-cell stage human embryos was recorded immediately before embryo transfer using a high-power inverted microscope. About 44% of patients (150/338) possessed embryos exhibiting MNB. The appearance of this nuclear abnormality was not correlated with maternal age. Overall, 15% of the otherwise good quality embryos (274/1885) that developed after monospermic fertilization contained several multinuclei (from two to seven) in at least one cell. Quite often MNB were found within all cells of the embryo (50% in 2-cell embryos). Blastomere multinucleation was significantly higher in 2-cell than 4-cell embryos (P <0.0001). This suggests that a considerable number of human embryos become abnormal during the first embryonic division. The embryos containing MNB were usually excluded for uterine transfers, with the exception of 19 cases when only such embryos could be replaced (6%; 19/338 patients). The results demonstrated that embryos with MNB may implant (4/19 cases; 21%) and they can lead to both spontaneous abortions and the successful birth of healthy infants (two cases). The fact that in the successful cases, 2-cell stage embryos with a mononucleated and a binucleated blastomere were transferred also suggests that due to the cell totipotency, development of a healthy baby is possible from one normal blastomere. Since multinucleation in early embryos may reflect gross chromosomal abnormalities or development of mosaic embryos, it is advisable not to replace embryos with MNB. Occasional transfers, however, can be considered because defective embryos may sometimes develop normally.      

The axis of polarity of the mouse blastocyst is specified before blastulation and independently of the zona pellucida

BACKGROUND: Rather than being prepatterned, orientation of the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst has been claimed to depend on the conceptus being constrained by its zona pellucida (ZP) during blastulation. This hypothesis merited closer scrutiny, because it seemed at variance with observations on living conceptuses. METHODS: Two-cell conceptuses with an oil drop injected into the lesser diameter (LD) of the ZP at the first cleavage plane were cultured until shortly before blastulation when the blastomere underlying the drop was labelled with carbocyanine dye. After removing the ZP, conceptuses were re-cultured to the blastocyst stage for recording the position along the axis of the centres of the patches of labelled cells. RESULTS: These centres showed significant bias towards the equatorial (Eq) region of the axis compared with those resulting from labelling a blastomere at random, even following softening of the ZP at the 2-cell stage. This was also true if conceptuses were denuded at the 2-cell stage and the blastomere underlying an intact second polar body (PB) labelled in morulae. CONCLUSIONS: These findings further support the view that the Em-Ab axis of the mouse blastocyst is normally prepatterned and provide no evidence of a role for the ZP in its specification.     

Human embryos with unevenly sized blastomeres have lower pregnancy and implantation rates: indications for aneuploidy and multinucleation

Uneven blastomere cleavage in human embryos of 'good morphology', i.e. those normally used for transfer, is a phenomenon which has been poorly investigated. The main objective in this study was to probe deeper into the aetiology behind previous findings that embryos with uneven cell cleavage have a lower developmental capacity in comparison with evenly cleaved embryos. Our hypothesis was that uneven cleavage may result in embryos with a higher degree of aneuploidy and/or multinuclear rate, which in turn might help to explain their low implantation rate. In the first part of the study, 378 embryo transfers performed over a 3-year period were analysed retrospectively, where all the transferred embryos in each cycle were of identical morphology score and cleavage stage. In the second part of the study, multicolour fluorescence in-situ hybridization (FISH) analyses on good quality embryos, representing the uneven (n = 11) and even (n = 13) study groups were performed. When comparing day 2 transfers between 4-cell embryos, it was found that unevenly cleaved embryos had significantly lower implantation (23.9 and 36.4%) and pregnancy rates (37.6 and 52.9%) compared with evenly cleaved embryos. A significantly higher degree of aneuploidy (29.4 and 8.5%) and multinuclear rate (21.1 and 2.1%) in blastomeres from uneven embryos was also found. It is concluded that uneven blastomere cleavage has a negative effect on both pregnancy and implantation rates in human IVF, and that this can partly be explained by a higher degree of aneuploidy/multinuclear rate. In the light of the results obtained, a new approach in the current embryo scoring system, placing more emphasis on blastomere size, is recommended.     

Surface-expressed E-cadherin, and mitochondrial and microtubule distribution in rescue of mouse embryos from 2-cell block by aggregation

E-cadherin (uvomorulin)-mediated cell interactions are essential for preimplantation development in mammals. We observed that E-cadherin is expressed at contact sites between blastomeres of 2-cell mouse embryos of non-blocking genotype (CBA x C57BL F1) explanted at 32 h post human chorionic gonadotrophin (HCG) and cultured in vitro, while blastomere rounding and reduced zones of contact and E-cadherin-staining were observed in embryos of a blocking strain (MF1) arrested at the 2-cell stage. Embryos of MF1 strain can be rescued by aggregation with four 2-cell embryos of the non-blocking genotype. An early event in rescue is E-cadherin expression at contact zones between adjacent embryos of different genotype in aggregation chimeras. E-cadherin-mediated signalling appears important for the rescue (including formation of adherens-like contacts, cell polarization and morphogenetic processes) since there is no rescue when E-cadherin-specific antibodies are present during phytohaemagglutinin-mediated aggregation and subsequent culture. In blocked embryos, the distribution of microtubules is disturbed and concomitantly mitochondria cluster around the nucleus. Rescue by aggregation retains normal mitochondrial distribution in the presence of a dense microtubular lattice in all blastomeres. Therefore, E-cadherin-mediated signalling and its downstream effects on cytoskeletal organization are essential in the rescue of blocking embryos by aggregation. Normal preimplantation development appears to be dependent on the polarized expression of surface E-cadherin and the microtubule-mediated dispersal of mitochondria.     

Role of Cdx2 and cell polarity in cell allocation and specification of trophectoderm and inner cell mass in the mouse embryo

Genesis of the trophectoderm and inner cell mass (ICM) lineages occurs in two stages. It is initiated via asymmetric divisions of eight- and 16-cell blastomeres that allocate cells to inner and outer positions, each with different developmental fates. Outside cells become committed to the trophectoderm at the blastocyst stage through Cdx2 activity, but here we show that Cdx2 can also act earlier to influence cell allocation. Increasing Cdx2 levels in individual blastomeres promotes symmetric divisions, thereby allocating more cells to the trophectoderm, whereas reducing Cdx2 promotes asymmetric divisions and consequently contribution to the ICM. Furthermore, both Cdx2 mRNA and protein levels are heterogeneous at the eight-cell stage. This heterogeneity depends on cell origin and has developmental consequences. Cdx2 expression is minimal in cells with unrestricted developmental potential that contribute preferentially to the ICM and is maximal in cells with reduced potential that contribute more to the trophectoderm. Finally, we describe a mutually reinforcing relationship between cellular polarity and Cdx2: Cdx2 influences cell polarity by up-regulating aPKC, but cell polarity also influences Cdx2 through asymmetric distribution of Cdx2 mRNA in polarized blastomeres. Thus, divisions generating inside and outside cells are truly asymmetric with respect to cell fate instructions. These two interacting effects ensure the generation of a stable outer epithelium by the blastocyst stage.     

Effect of phosphate on the second cleavage division of the rat embryo

Development of the rat embryo is arrested at the 2-cell stage in vitro in the presence of inorganic phosphate (Pi). Rat embryos were affected by exposure to 1.19 mM KH2PO4 in modified hamster embryo culture medium- 1 at the late 2-cell stage only. When exposure durations were 6 h, embryos whose exposure timings were prior to cleavage had a reduced rate of development to the blastocyst stage (2-8%) when compared with embryos with no exposure to Pi (97%, P < 0.05). When exposure durations were 18 h, all embryos were arrested at the 2- to 4-cell stage. These timings would correspond to the G2 to M phase of the second cell cycle. Maturation-promoting factor (MPF), which is regulated by a phosphorylation cascade, controls cell division, and its kinase activity is necessary in order for the cell to enter the M phase. However, the histone H1 kinase activity levels and the patterns of the state of phosphorylation of cdc2 were the same in blocked and non- blocked embryos. Because MPF was active in blocked embryos, the developmental block in rat 2-cell embryos caused by phosphate was not due to MPF activity or its phosphorylation cascade.      

Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development

BACKGROUND: It is of fundamental importance for IVF clinics to determine the most viable embryos for transfer. The challenge for ART clinics is to transfer fewer embryos, thereby minimizing the risk of multiple-infant births, while still maintaining the greatest chance of pregnancy for their patients. In this study, an investigation was made to determine if developmental markers on the day of fertilization (day 1) can predict good subsequent blastocyst development. METHODS AND RESULTS: A total of 1550 individually cultured 2PN embryos from 191 patients undergoing IVF/ICSI treatment at the Yale University Center for Reproductive Medicine and Infertility from February to December 2001 was included. The results showed a significant positive relationship between early-cleaving 2-cell embryos and subsequent good quality > or =4-cell, > or =7-cell and blastocyst development (P < 0.05). PN symmetry (the relative size of the PN to each other), when checked at the time fertilization, is also a significant indictor of good quality > or =4-cell, > or =7-cell stage embryos and blastocysts. Combined, a developing embryo showing PN symmetry with early cleavage and subsequent good > or =4-cell and > or =7-cell cleavage, has a one in two chance of developing into a good-quality blastocyst. CONCLUSION: Early embryo assessment can be used as an indicator of subsequent good blastocyst development.     

Morphometric categorization of the human oocyte and early conceptus

Morphometrical procedures were used to quantitatively evaluate human oocytes and embryos in an IVF programme. The metaphase II oocyte was an irregular 3.5 x 10(6) microns 3 sphere of 1.05 coefficient of form. The ooplasmic volume of 1.4 x 10(6) microns 3 was reduced by 10% by fertilization. The zona pellucida behaved as a stable and almost spherical envelope of 1.8 x 10(6) microns 3 volume and 17 microns thickness. Through the first three cleavages, mean blastomere reduces 28.5% volume per division, evolving from an irregular spherical shape with 0.9 coefficient of form to an ellipsoid (0.8) at the 8-cell stage. The coefficient of diversity between sister blastomeres progressively moved from 1.4 to 1.6 during the first two (2n) cleavages. The coefficient of diversity also increased at 3-cell (2.2) and 6-cell (2.6) asynchronous divisions. Morphologically abnormal embryos showed some morphometrical differences. Embryos which successfully implanted and progressed to birth showed a higher coefficient of diversity between sister blastomeres.     

A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos

The impact of cryopreservation on the implantation potential of early cleavage stage (day 2) embryos was assessed by analysing the outcome from > 5000 thawed embryos in relation to the outcome from a similar number of fresh embryos. Analysis of procedures in which all transferred embryos fulfilled equivalent defined criteria revealed no significant difference in the implantation rates (fetal hearts/100 embryos transferred) of fresh 4-cell embryos (16.6%) and fully intact thawed 4-cell embryos (16.9%). Although 2-cell embryos implanted at significantly lower rates, there was again no significant difference between fresh (6.5%) and fully intact thawed (7.2%) embryos. Similar analysis of all embryos (irrespective of cell number on day 2) demonstrated that the implantation potential of partially intact thawed embryos was related to the extent of blastomere loss with the implantation rate of embryos with 50% cell survival (5.4%) being approximately half the rate of fully intact embryos (11.3%). Combining the values obtained from 'pure' data for the implantation rates of embryos with defined levels of survival with their relative prevalence in the total population of thawed embryos gave a predicted number of implantations (441) which was similar to the observed outcome (463). This number was approximately 30% less than the number expected had the same embryos been transferred fresh (635). The results suggest that intact thawed embryos have the same implantation potential as equivalent fresh embryos and that the impact of cryopreservation is limited to blastomere loss which is directly related to loss of implantation potential. The observed frequency of blastomere loss results in a reduction of approximately 30% in the implantation potential of a population of embryos following cryopreservation.     

Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence

It has been suggested that mitochondrial DNA defects that effect metabolic capacity may be a proximal cause of failures in oocyte maturation, fertilization, or early embryonic development. Here, the distribution of mitochondria was examined by scanning laser confocal microscopy in living human pronuclear oocytes and cleavage stage embryos, followed either by measurements of the net ATP content of individual blastomeres or anti-tubulin immunofluorescence to determine the relationship between mitochondrial distribution and microtubular organization. The results indicate that specific patterns of perinuclear mitochondrial aggregation and microtubular organization are related, and that asymmetrical mitochondrial distributions at the pronuclear stage can result in some proportion of blastomeres with reduced mitochondrial inheritance and diminished ATP generating capacity. While the inability to divide appears to be a development consequence for an affected blastomere, for the embryo, reduced competence may occur during cleavage if several blastomeres inherit a mitochondrial complement inadequate to support normal cellular functions. The findings provide a possible epigenetic explanation for the variable developmental ability expressed within cohorts of morphologically normal early cleavage stage human embryos obtained by in-vitro fertilization.     

Ultrastructural and autoradiographic studies of mouse cleavage stages

Studies of cleavage stage mouse embryos are reported, with particular emphasis upon nucleolar fine structural and functional changes. Multiple fibrillar primary nucleoli are present in the early 2-cell embryo. In late 2-cell embryos, some of these nucleoli acquire a peripheral zone of granules, while others reticulate, forming nucleoli composed of fibrillo-granular cortices and fibrillar cores. The nucleoli of early 4-cell embryos are composed only of fibrils. In the middle of the 4-cell stage, some of the nucleoli acquire a peripheral granular zone, while others reticulate. The reticulated nucleoli of both the late 2-cell and 4-cell embryos can be considered, on the basis of their fine structure, to be definitive nucleoli. Early 8-cell and morula embryos usually contain only two definitive nucleoli per nucleus. ³H-5-uridine-pulsed embryos contain label localized in the nucleus, particularly over definitive nucleoli. Nucleolar labeling increases at each successive developmental stage. Beginning at the 8-cell stage, re-incubation in nonradioactive medium results in a significant decrease in nucleolar labeling and an increase in cytoplasmic labeling suggesting that more ribosomal RNA is transferred from the nucleus to the cytoplasm at the later cleavage stages.     

Ploidy in human cleavage stage embryos after fertilization in vitro

Chromosome preparations were made from 15 cleaved human embryosin the 2- to 12-cell stage after in-vitro fertilization. Allshowed two pronuclei before the first cleavage. Twelve had atleast one diploid metaphase, while three had interphase nucleionly. There was no evidence of partheno-genetic haploid cleavage.As expected, the frequency of metaphases increased with durationof colchicine treatment: 25 and 55% of the cells reached metaphaseafter 6–13 and 16–24 hours' treatment, respectively.Cleaved embryos with ‘ideal’ blastomere numbers(2, 4 or 8) showed a considerably higher metaphase frequencythan others. For a more detailed chromosome analysis the techniquewill have to be further improved. The asynchronous cleavageof blastomeres makes optimal treatment by mitogens difficult.     

The genetic constitution of multinuclear blastomeres and their derivative daughter blastomeres

The presence of multinuclear blastomeres (MNB) has been widely reported in in-vitro-cultured embryos. Multinucleation at the first mitotic division and affecting both blastomeres is considered abnormal and such embryos are not transferred. The objective of this study was to use fluorescent in-situ hybridization (FISH) and probes specific for chromosomes X, Y and 18 to examine the genetic constitution of embryos developing from the 2-cell stage in which both blastomeres were bi- or multinuclear. Initially, 2-cell embryos in which both blastomeres were bi- or multinuclear were cultured further. Of 101 embryos, 89 (88.1%) cleaved further and were analysed at the 3- to 8-cell stage on day 2 or 3. Among embryos analysed, 30.4% contained only mononuclear diploid blastomeres, 35.9% had a combination of mononuclear diploid and non- diploid blastomeres, and 33.7% had non-diploid blastomeres, indicative of chaotic division. Results obtained were similar with embryos derived from in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Also, no significant differences were found between 2-cell embryos with bi- or multinuclear blastomeres or between slowly or normally cleaved embryos. Twelve (11.9%) embryos arrested at the 2-cell stage on day 3; of these, one had diploid blastomeres and the others were abnormal and highly polyploid. Subsequently, 59 embryos were analysed at the 2-cell stage. Initial observations related to the high number of nuclei in metaphase at the moment of spreading, notably when multinuclear blastomeres were observed. Genetic analysis showed 44.7% of embryos to be susceptible to analysis; the genetic constitution corresponded in both blastomeres to a diploid status. A combined diploid blastomere and abnormal blastomere was found in 4.3% of embryos; both blastomeres were abnormal in 51%. These data show that the genetic constitution of bi- or multinuclear blastomeres, and the daughter cells developing from them, are not always abnormal.      

小鼠2-细胞胚胎卵裂球后代在囊胚中随机分布

目的 用异硫氰荧光素（FITC）-右旋糖苷和四甲基罗丹明（TMR）-右旋糖苷标记小鼠2-细胞胚胎的两个卵裂球,观察其发育,以探讨囊胚Em-Ab极性的形成。方法 向受精卵注射FITC-右旋糖苷确定标记物对胚胎的伤害性,向2-细胞胚胎两个卵裂球分别FITC-右旋糖苷分别和TMR-右旋糖苷,将标记后的胚胎体外培养发育至囊胚,观测两个卵裂球后代在囊胚中的分布情况。 结果 2-细胞胚胎两个卵裂球的后代在囊胚中分布并无规律。 结论 2-细胞胚胎卵裂球随机分布在囊胚的胚胎部分和胚外部分。