155 CHARACTERIZATION OF LIPID DROPLETS IN BOS INDICUS AND BOS TAURUS EMBRYOS

Accurate evaluation of bovine embryos for assessing developmental stage and quality is critical to the success of any embryo transfer program. However, this evaluation process has been reported to be highly subjective in Bos indicus (BI) and can vary as much as 23% compared with that of Bos taurus (BT). These differences in assessment may be related to the quantity of lipid droplets (LD) within the embryo, which has been shown to have a negative effect in cryopreserving embryos. The aim of the present study was to characterize the number and size of LD in different developmental stages of fresh embryos from BI and BT and to compare LD across the three different embryo quality grades (1 = excellent or good, 2 = fair, and 3 = poor). Nonsurgical embryo collection was performed 7 days post-insemination in 10 BI and 10 BT females. Forty-eight embryos were evaluated for stage and grade using stereoscopic microscopy, processed for transmission electron microscopy, and stained with Nile red. Digitalized images were analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA), contour of lipid droplets were designed, and values of perimeter, area, and fluorescence intensity were assessed. Nonparametric statistical analysis (Mann–Whitney) was utilized. There was no difference in LD number for BT or BI for morulae and blastocyst; however, BI morulae presented larger LD compared with blastocyst stage embryos (286 µm2 v. 223 µm2; P < 0.05). Likewise, BI TF cells had more LD compared with inner cell mass (ICM) cells (48 v. 36; P < 0.05). BT TF cells exhibited larger LD compared with ICM cells (149 µm2 v. 128 µm2; P < 0.05), while BI embryos exhibited a larger area of LD in the ICM compared with the TF (591 µm2 v. 472 µm2; P < 0.05). In all embryos, BI contained more lipid droplets than BT (78 v. 49; P < 0.05). Across all quality grades (good, fair, and poor) there was no difference in the number of LD in BT embryos; however, BI grade-3 embryos presented more LD than grade-1 (36 v. 25). BT embryos LD were larger than BI LD (907 µm2 v. 625 µm2; P < 0.05). Fluorescence images showed higher arbitrary units of fluorescence (auf) for LD in BI. Compared with BT embryos (386 auf v. 280 auf; P < 0.05). These results suggest that BI embryos contain more and smaller LD than BT embryos and the LD described for BI embryo quality grade 1 are larger than those of quality grades 2 and 3, and even though the number of LD in morulae and blastocyst stage embryos are not different LD size is reduced as development occurs. Research funding provided by UNAM-DGAPA-PAPIIT IN200810.

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61 DIPLOID (BOS TAURUS) AND TETRAPLOID (BOS INDICUS) EMBRYO AGGREGATION TO PRODUCE BOVINE EMBRYONIC CHIMERAS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab61 ◽

2012 ◽

Vol 24 (1) ◽

pp. 142

Author(s):

E. Montanari Razza ◽

R. A. Satrapa ◽

I. P. Emanuelli ◽

C. Moraes Barros ◽

M. F. G. Nogueira

Keyword(s):

Bos Taurus ◽

Inner Cell Mass ◽

Cell Mass ◽

Bos Indicus ◽

Single Pulse ◽

Blastocyst Stage ◽

Random Pattern ◽

Cell Stage ◽

Protease Treatment ◽

Holstein Bulls

The formation of tetraploid embryos (4n) by electrofusion and their subsequent chimerism with a diploid embryo (2n) must result in a chimeric conceptus, whose inner cell mass (ICM) is entirely 2n. Hence, the aggregation of a zebu embryo (4n, thermotolerant) with a taurine embryo (2n, thermosensitive) would result in an exclusively taurine ICM, but the trophectoderm (future extraembryonic components) would be mostly from the zebu embryo, which could interact with the taurine embryo/fetus in a different way (than it occurs in whole Bos taurus conceptus) during pregnancy in a tropical environment. The purpose of this study was to standardize the production of 4n Nelore embryos (Bos indicus) and the production of embryonic chimeras by aggregation of 1/2 B. taurus (2n) with B. indicus (4n) embryos. Oocytes from Nelore cows from the abattoir were matured, fertilized with semen from Nelore and Holstein bulls and cultured in SOF. Two-cell stage Nelore embryos (30 h post-insemination), with a well-defined inter-blastomeric axis, were selected for the electrofusion procedure (ECM 830-BTX, Harvard Apparatus) to produce 4n embryos. For this procedure, some parameters were tested according to the number of pulses (1 or 2), voltage (40, 50, 75, 100, 140 and 500 V) and duration of electroshock (20, 25, 50 and 60 μs). Nelore 4n embryos produced after electrofusion and 2n taurine embryos, both at 8 to 16-cell stages (72 h post-insemination) were subjected to protease treatment to remove the zona pellucida and subsequently treated with the agglutinant agent phytohemagglutinin. Bos indicus (4n) and B. taurus (2n) embryos were added in pairs (4n+2n) into individual wells (well of the well, WOW) for culture until the blastocyst stage to validate the chimeric embryos formation. Among the tested parameters, the best fusion results (92%) and rates of cleavage after fusion (66%) were obtained with a single pulse of 75 V for 60 μs. The production rate of expanded 4n blastocysts was 31.5% using these parameters. After 4 replicates (still in the final stage of standardization), 4 blastocyst chimeras (4n+2n) were obtained from 31 attempts (13%). The production of bovine embryonic chimeras [Bos indicus (4n) + Bos taurus (2n)], with a non-random pattern of distribution of their cell aggregates, will enable the validation of this technique in applied research, by producing exclusively taurine calves, but with placental elements from the B. indicus breed, following transfer of these chimeras into recipient cows. Financial support received from FAPESP, Brazil.

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129 KNOCKDOWN OF WBP1 DECREASES TROPHECTODERM FORMATION IN THE PRE-IMPLANTATION BOVINE EMBRYO

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab129 ◽

2017 ◽

Vol 29 (1) ◽

pp. 173

Author(s):

M. S. Ortega ◽

P. J. Hansen

Keyword(s):

Embryonic Development ◽

Bos Taurus ◽

Inner Cell Mass ◽

Cell Mass ◽

Bos Indicus ◽

Cell Number ◽

Blastocyst Stage ◽

Fold Change ◽

Bovine Embryo

A single nucleotide polymorphism (SNP) in WBP1 has been previously associated with embryonic development to the blastocyst stage. WBP1 interacts with WW domain containing proteins including YAP1 from the hippo signalling pathway that is involved in trophectoderm (TE) formation. Here we tested whether reduction in mRNA abundance for WBP1 would reduce development to the blastocyst stage and formation of cells in the inner cell mass (ICM) and TE. Knockdown was performed using a GapmeR LNATM antisense oligonucleotide designed to target WBP1. A scrambled version of the same sequence was used as a control. Embryos were produced in vitro from slaughterhouse oocytes and bulls from Bos taurus and Bos indicus breeds. At 20 to 22 h after insemination (hpi), embryos were treated with 5 µM anti-WBP1 GapmeR (KD), 5 µM scrambled GapmeR (SC), or vehicle (CTL). At 72 to 75 hpi (the time of maximal WBP1 expression), groups of 18 to 20 embryos were collected from each treatment to evaluate WBP1 expression. Other cultured embryos (minimum of 50/treatment for each replicate) were cultured until Day 8 after insemination. Cleavage was assessed at Day 3 and blastocyst formation at Day 7 and 8. Embryos were collected at Day 8 to determine ICM and TE cell number by determining nuclear immunoreactive CDX2. All experiments were replicated 5 times. Fold change was calculated relative to the CTL group. Data were analysed by analysis of variance for gene expression and cell number, and through logistic regression for embryonic development. WBP1 expression was reduced (P = 0.04) in KD embryos compared to CTL (least squares means ± SEM: 1 ± 0.19 v. 0.64 ± 0.19 fold change) or SC (1.05 ± 0.19). There was no difference in expression between CTL and SC. Percent of embryos that cleaved was not affected by treatment (P > 0.05); however, percent of inseminated oocytes that became blastocysts tended to be lower in KD compared to CTL and SC at Day 7 (P = 0.09) [10.8 ± 2.8, 20 ± 3.0, and 16.3 ± 3.1% for KD, CTL, and SC, respectively] and 8 after insemination (P = 0.06) [13.7 ± 3.3, 24.2 ± 3.3, and 22.9 ± 3.6%]. Knockdown of WBP1 caused a reduction in number of total (P = 0.0004) and TE (P < 0.0001) cells with no effect on ICM cell number (P = 0.83). Total cell numbers for KD, SC, and CTL were 124.2 ± 6.4, 157.75 ± 7.4, and 124.28 ± 6.4 and numbers of TE cells were 59.7 ± 3.8, 90.0 ± 4.47, and 90.0 ± 4.4. Results show that reduction in mRNA for WBP1 decreases TE formation and tends to reduce competence of embryos to become blastocysts. This study was supported by USDA AFRI 2013–68004–20365.

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Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development

Development ◽

10.1242/dev.115.1.305 ◽

1992 ◽

Vol 115 (1) ◽

pp. 305-312

Author(s):

M. Aghayan ◽

L.V. Rao ◽

R.M. Smith ◽

L. Jarett ◽

M.J. Charron ◽

...

Keyword(s):

Western Blot ◽

Cellular Localization ◽

Glucose Transporter ◽

Developmental Stages ◽

Molecular Level ◽

Inner Cell Mass ◽

Cell Mass ◽

Blastocyst Stage ◽

Preimplantation Development ◽

Developmental Expression

Two general mechanisms mediate glucose transport, one is a sodium-coupled glucose transporter found in the apical border of intestinal and kidney epithelia, while the other is a sodium-independent transport system. Of the latter, several facilitated transporters have been identified, including GLUT1 (erythrocyte/brain), GLUT2 (liver) and GLUT4 (adipose/muscle) isoforms. In this study, we used Western-blot analysis and high resolution immunoelectron microscopy (IEM) to investigate the stage-related expression and cellular localization of GLUT1, 2 and 4. The Western blot results demonstrate that GLUT1 is detectable in the oocyte and throughout preimplantation development. GLUT2 isoforms were not detectable until the blastocyst stage, while the GLUT4 isoform was undetectable in the oocyte through blastocyst stages. The present findings confirm previous studies at the molecular level which demonstrated that mRNAs encoding the same GLUT isoforms are detectable at corresponding developmental stages. GLUT1 and GLUT2 display different cellular distributions at the blastocyst stage as shown by IEM studies. GLUT1 has a widespread distribution in both trophectoderm and inner cell mass cells, while GLUT2 is located on trophectoderm membranes facing the blastocyst cavity. This observation suggests a different functional significance for these isoforms during mouse preimplantation development.

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Ultrastructural observations of lethal yellow (Ay/Ay) mouse embryos

Development ◽

10.1242/dev.35.1.73 ◽

1976 ◽

Vol 35 (1) ◽

pp. 73-80

Author(s):

Patricia G. Calarco ◽

Roger A. Pedersen

Keyword(s):

Developmental Stages ◽

Inner Cell Mass ◽

Structural Characteristics ◽

Cell Mass ◽

Blastocyst Stage ◽

Cell Organelles ◽

Cell Stage ◽

Wide Range ◽

Lethal Yellow ◽

And Control

Ay/Ay embryos were identified by the presence of large excluded blastomeres (Pedersen, 1974) and examined cytologically and ultrastructurally. Cell organelles, inclusions and junctions in the excluded blastomeres were compared with those of non-excluded cells of Ay/Ay embryos and control embryos. Excluded blastomeres always had the fine structural characteristics of earlier developmental stages and may have arrested at the 4- to 8-cell stage or slightly later. Interior cells (inner cell mass) were observed in all mutant blastocysts. Nonexcluded cells of Ay/Ay embryos were normal until degenerative changes appear in the late blastocyst stage. The mode of action of the +Ay gene was not determined, but evidence from this study and others indicates that the effects of +Ay gene action occur over a wide range of time in early cleavage and implantation.

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In vitro production of cattle-water buffalo (Bos taurus - Bubalus bubalis) hybrid embryos

Zygote ◽

10.1017/s0967199402002216 ◽

2002 ◽

Vol 10 (2) ◽

pp. 155-162 ◽

Cited By ~ 13

Author(s):

H.P.S. Kochhar ◽

K.B.C. Appa Rao ◽

A.M. Luciano ◽

S.M. Totey ◽

F. Gandolfi ◽

...

Keyword(s):

Water Buffalo ◽

Bos Taurus ◽

Inner Cell Mass ◽

Cell Mass ◽

Bubalus Bubalis ◽

Blastocyst Stage ◽

In Vitro Production ◽

Cleavage Rate ◽

Developmental Potential

Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using interspecies gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.

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The second lineage differentiation of bovine embryos fails in the absence of OCT4/POU5F1

10.1101/2021.09.06.459107 ◽

2021 ◽

Author(s):

Kilian Simmet ◽

Mayuko Kurome ◽

Valerie Zakhartchenko ◽

Horst-Dieter Reichenbach ◽

Claudia Springer ◽

...

Keyword(s):

Developmental Stages ◽

Inner Cell Mass ◽

Ex Vivo ◽

Expression Patterns ◽

Cell Mass ◽

Blastocyst Stage ◽

Bovine Embryos ◽

Vitro Fertilization ◽

Lineage Differentiation

The mammalian blastocyst undergoes two lineage segregations, i.e., formation of the trophectoderm and subsequently differentiation of the hypoblast (HB) from the inner cell mass, leaving the epiblast (EPI) the remaining pluripotent lineage. To clarify expression patterns of markers specific for these lineages in bovine embryos, we analyzed day 7, 9 and 12 blastocysts completely derived ex vivo by staining for OCT4, NANOG, SOX2 (EPI) and GATA6, SOX17 (HB) and identified genes specific for these developmental stages in a global transcriptomics approach. To study the role of OCT4, we generated OCT4-deficient (OCT4 KO) embryos via somatic cell nuclear transfer or in vitro fertilization. OCT4 KO embryos reached the expanded blastocyst stage by day 8 but lost of NANOG and SOX17 expression, while SOX2 and GATA6 were unaffected. Blastocysts transferred to recipient cows from day 6 to 9 expanded, but the OCT4 KO phenotype was not rescued by the uterine environment. Exposure of OCT4 KO embryos to exogenous FGF4 or chimeric complementation with OCT4 intact embryos did not restore NANOG or SOX17 in OCT4-deficient cells. Our data show, that OCT4 is required cell-autonomously for the maintenance of pluripotency of the EPI and differentiation of the HB in bovine embryos.

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244 CHANGES IN THE TRANSCRIPTOME OF THE BOVINE PRE-IMPLANTATION EMBRYO INDUCED BY COLONY-STIMULATING FACTOR-2

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab244 ◽

2010 ◽

Vol 22 (1) ◽

pp. 279

Author(s):

B. Loureiro ◽

L. Oliveira ◽

P. J. Hansen

Keyword(s):

Bos Taurus ◽

Inner Cell Mass ◽

Transcriptional Profiling ◽

Cell Mass ◽

Blastocyst Stage ◽

Differentially Expressed ◽

Colony Stimulating Factor ◽

Apoptosis Pathways ◽

Stimulating Factor

Colony-stimulating factor-2 (CSF-2) is a cytokine expressed in bovine oviduct and endometrium that has been reported to improve the proportion of embryos that become blastocysts in vitro and survive after transfer to recipients. One effect of CSF-2 that might be related to increased embryonic survival is a preferential increase in the number of cells in the inner cell mass. The objective of the current study was to determine changes in the embryo transcriptome caused by CSF-2 that promote blastocyst formation and establishment and maintenance of pregnancy after transfer. Bovine embryos were produced in vitro and cultured in KSOM-BE2 +10 ng/mL recombinant BoCSF-2 added at Day 5 after insemination. On Day 6 (24 h after treatment), embryos at the morula and early blastocyst stage were harvested and stored in groups of 50 at -80°C. A total of 4 pools of GM-colony-stimulating factor treated blastocysts and 4 control blastocysts were subjected to transcriptional profiling using the Bos taurus 2-color Agilent chip (4 × 44 K format). Before labeling, total RNA starting sample was spiked with control genes (artificial clones) of known concentration provided by Agilent. Labeling was done simultaneously with complimentary RNA (cRNA) amplification. Two rounds of linear RNA amplification were employed. Images were extracted using the Agilent Feature Extraction Software (Agilent Technologies, Santa Clara, CA, USA) and normalized within arrays by the Lowess method. Statistical analysis was performed using the JMP Genomics program (SAS Inst., Cary, NC, USA). The normalized data were log2 transformed, and the quantile normalization method was used to normalize data between arrays. Differences in gene expression were determined using PROC ANOVA (fixed false discovery rate = 0.01). Only genes with a 1.5-fold difference and P < 0.05 were considered differentially expressed. A total of 216 genes were differentially expressed between CSF-2 and control embryos. Of these, 141 could be annotated (61 genes up-regulated and 80 genes down-regulated by CSF-2). These included 13 genes involved in Wnt pathways, including 5 inhibitors of Wnt signaling (FRP, MAB21L2, PCDH24, PDE7, PPPR23A) that were up-regulated by CSF-2 and 5 genes involved in transmission of Wnt signals (WNT16, ROR2, CSNK2B, CELSR2, DTX3) that were down-regulated by CSF-2. Several other genes associated with differentiation were down-regulated by CSF-2 including CXCL12, FEZF1, PLD2, and RGS12. Expression of 1 gene that inhibits apoptosis (PRKAR2B) was increased by CSF-2, whereas expression of 6 genes involved in apoptosis pathways (DAPK1, MADD, NOD2, PIK3IP1, RIPK3, RNF7) were down-regulated. Results indicate that CSF-2 promotes pluripotency and decreases apoptosis in bovine pre-implantation embryos. This research was supported by USDA-AFRI. B. Loureiro andL. Oliveira were supported by a CAPES (Brazil)/Fulbright Fellowship.

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P–259 Blastocyst quality is associated with both the start and the duration of compaction after ICSI

Human Reproduction ◽

10.1093/humrep/deab130.258 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

K Wouters ◽

L Va. Landuyt ◽

M Regin ◽

H Tournaye ◽

G Verheyen ◽

...

Keyword(s):

Embryo Quality ◽

Inner Cell Mass ◽

Cell Mass ◽

Total Duration ◽

Time Lapse ◽

Poor Quality ◽

Blastocyst Stage ◽

Human Embryos ◽

The Poor ◽

Blastocyst Quality

Abstract Study question Is the start and the total duration of compaction related to embryo quality? Summary answer The timing of the start, the end and the total duration of compaction are associated with blastocyst quality grade in the IVF laboratory. What is known already Preimplantation embryo development follows a programmed timeline during which a series of critical events take place. One event typically occurring on day 3/4 post fertilisation is the formation of adherence junctions between blastomeres in a process called compaction. It is considered the first morphological event in the differentiation process of the mammalian embryo. Evaluation of developmental events are used to optimize the selection of the most competent embryos for transfer and/or cryopreservation in the IVF laboratory. It has already been shown that the time of full compaction is indicative for high-quality blastocysts with a higher implantation rate. Study design, size, duration A single-centre retrospective observational study including 74 ICSI cycles performed in 2020. Injected oocytes were cultured in blastocyst medium (Origio) in the EmbryoScope + (Vitrolife) for 5/6 days. Embryos that reached the blastocyst stage were evaluated for the start of compaction, the time to reach full compaction and the total duration of compaction. These parameters were compared between good- and poor-quality blastocysts; the primary outcome parameter of the study was embryo quality. Participants/materials, setting, methods Only ICSI cycles with ejaculated fresh/frozen-thawed sperm and monitored in time-lapse incubator were included. All MNC, IVM and PGT cycles were excluded. Time zero was the start of ICSI. Good-quality embryos were full and expanded blastocysts with good-quality inner cell mass and trophectoderm (AA, AB, BA and BB according to Gardner and Schoolcraft (1999)). GraphPad Prism was used for statistical analysis. After testing for normality and homogeneity, unpaired t-test or Mann-Whitney test determined significant differences. Main results and the role of chance In this study, of the 528 included 2PN oocytes, 229 (43.4%) reached the blastocyst stage and 299 (56.6%) were arrested. Among the former, 131 (57.2%) blastocysts were classified in the good-quality group and 98 (42.8%) blastocysts in the poor-quality group. In general, human embryos compacted slowly while dividing further and the blastomeres moved during the compaction process. The start of compaction was heterogeneous (between 50.9 and 102.7 hours post ICSI; mean=80.0 hours), as well as the cell number at the initiation (between 4 and 18 blastomeres; mean=12 blastomeres). The time analysis showed that the embryos in the good-quality group started to compact significantly earlier than those in the poor-quality group (mean=78.6 vs 82.2 hours; R²=0.06; p < 0.01). We confirmed that blastocysts in the good-quality group reached full compaction earlier than those in the poor-quality group (mean=86.8 vs 93.8 hours; R²=0.17; p < 0.01). Furthermore, the total duration of compaction was significantly lower in the good-quality than in the poor-quality group (median=7.4 vs 10.7 hours; p < 0.01). Limitations, reasons for caution As this is a retrospective study, the influence of uncontrolled variables cannot be excluded. The absence of the pregnancy outcome and live birth rate is a shortcoming and will be subject of a larger patient-to-patient study. Wider implications of the findings: These results indicate that an earlier start and a shorter duration of compaction are associated with better blastocyst quality. These morphological events can be valuable additional parameters in selecting the embryo of better quality when using a time-lapse incubator. Trial registration number Not applicable

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Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo

eLife ◽

10.7554/elife.22906 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 55

Author(s):

Eszter Posfai ◽

Sophie Petropoulos ◽

Flavia Regina Oliveira de Barros ◽

John Paul Schell ◽

Igor Jurisica ◽

...

Keyword(s):

Gene Expression ◽

Inner Cell Mass ◽

Cell Mass ◽

Global Gene Expression ◽

Blastocyst Stage ◽

Egfp Expression ◽

Hippo Signaling ◽

Ability To Regenerate ◽

Early Mouse ◽

Cell Commitment

The segregation of the trophectoderm (TE) from the inner cell mass (ICM) in the mouse blastocyst is determined by position-dependent Hippo signaling. However, the window of responsiveness to Hippo signaling, the exact timing of lineage commitment and the overall relationship between cell commitment and global gene expression changes are still unclear. Single-cell RNA sequencing during lineage segregation revealed that the TE transcriptional profile stabilizes earlier than the ICM and prior to blastocyst formation. Using quantitative Cdx2-eGFP expression as a readout of Hippo signaling activity, we assessed the experimental potential of individual blastomeres based on their level of Cdx2-eGFP expression and correlated potential with gene expression dynamics. We find that TE specification and commitment coincide and occur at the time of transcriptional stabilization, whereas ICM cells still retain the ability to regenerate TE up to the early blastocyst stage. Plasticity of both lineages is coincident with their window of sensitivity to Hippo signaling.

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The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽

10.1242/dev.107.3.597 ◽

1989 ◽

Vol 107 (3) ◽

pp. 597-604 ◽

Cited By ~ 13

Author(s):

K. Hardy ◽

A.H. Handyside ◽

R.M. Winston

Keyword(s):

Cell Death ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Blastocyst Stage ◽

Human Embryos ◽

Cell Numbers ◽

Human Blastocyst ◽

Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

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