41 Delineating the molecular connections between mitotic aneuploidy, micronucleation, and cellular fragmentation in pre-implantation bovine embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 146
Author(s):  
K. E. Brooks ◽  
B. L. Daughtry ◽  
S. S. Fei ◽  
M. Y. Yan ◽  
B. Davis ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Rna Sequencing ◽  
Live Cell ◽  
Confocal Imaging ◽  
Chromosomal Mosaicism ◽  
Human Embryos ◽  
Bovine Embryos ◽  
Cleavage Stage ◽  
Cell Stage

Whole chromosomal abnormalities (aneuploidy) that arise during early embryo development are a major contributor to in vitro fertilization failure. It is estimated that ~50 to 80% of human embryos contain aneuploid cells, which contribute to high levels of chromosomal mosaicism detected by pre-implantation genetic screening. Previous studies estimate that 32 to 88% of bovine embryos are aneuploid at the 2-cell stage, advocating cattle as a physiologically relevant model to study the mechanisms mediating meiotic and/or mitotic errors. In cleavage-stage human embryos, a process called cellular fragmentation is associated with aneuploidy, and when used in conjunction with assessment of early mitotic timing, can largely distinguish chromosomally normal and abnormal embryos. We recently demonstrated that some cellular fragments contain chromosomal material that likely began as mis-segregated chromosomes that were encapsulated into micronuclei. Given that bovine embryos exhibit cellular fragmentation, albeit to a lesser extent than human embryos, we hypothesise that cellular fragmentation is a response to micronucleation and represents a conserved mechanism to eliminate mis-segregated chromosomes from the pre-implantation embryo. Using a combination of live-cell imaging, single-cell DNA-sequencing, whole-embryo RNA-sequencing, quantitative RT-PCR, and multicolour confocal microscopy, we aim to further investigate the correlation between these phenomena using in vitro-produced bovine embryos. Similar to humans, the first three mitotic divisions are able to successfully predict progression to the blastocyst stage (N=84). Bovine embryos frequently contained multi-/micro-nuclei, and DNA-sequencing of individual bovine blastomeres up to 12 cells confirmed that ~58 to 87% of cleavage-stage bovine embryos are aneuploidy (N=38) and often detectable by abnormal cell divisions. Transcriptional profiling of fragmented versus non-fragmented bovine embryos via RNA-sequencing identified a small subset of differentially abundant genes at the 4-cell stage. Pathway analysis showed reduced abundance of genes associated with the cytoskeleton, microtubules, and spindle in 4-cell embryos with cellular fragmentation as well as enrichment of membrane targeting and vesicle fusion pathways. The potential role of these cellular components in micronucleation and cellular fragmentation is being assessed by microinjecting bovine zygotes with fluorescently labelled mRNA mCherry-H2B (chromatin marker) and mCitrine-LaminB1 (nuclear envelope marker), followed by overnight live-cell multicolour confocal imaging (Zeiss LSM 880 with AiryScan; Zeiss, Thornwood, NY, USA). Results from these studies contribute to our knowledge of early embryogenesis with translational application to help ameliorate embryonic loss in women and cattle.

6 ANEUPLOIDY TOLERANCE IN RHESUS MACAQUE PRE-IMPLANTATION EMBRYOS VIA MICRONUCLEI FORMATION, CELLULAR FRAGMENTATION, AND BLASTOMERE EXCLUSION

2017 ◽  
Vol 29 (1) ◽  
pp. 110 ◽  
Author(s):  
B. L. Daughtry ◽  
J. L. Rosenkrantz ◽  
N. Lazar ◽  
N. Redmayne ◽  
K. A. Nevonen ◽  
...  
Keyword(s):  
Rhesus Macaque ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Chromosomal Mosaicism ◽  
Human Embryos ◽  
Cleavage Stage ◽  
Chromosomal Breakage

A primary contributor to in vitro fertilization (IVF) failure is the presence of unbalanced chromosomes in pre-implantation embryos. Previous array-based and next-generation sequencing (NGS) studies determined that ~50 to 80% of human embryos are aneuploid at the cleavage stage. During early mitotic divisions, many human embryos also sequester mis-segregated chromosomes into micronuclei and concurrently undergo cellular fragmentation. We hypothesised that cellular fragmentation represents a response to mis-segregated chromosomes that are encapsulated into micronuclei. Here, we utilised the rhesus macaque pre-implantation embryo as a model to study human embryonic aneuploidy using a combination of EevaTM time-lapse imaging for evaluating cell divisions, single-cell/-fragment DNA-Sequencing (DNA-Seq), and confocal microscopy of nuclear structures. Results from our time-lapse image analysis demonstrated that there are considerable differences in the timing of the first and third mitotic divisions between rhesus blastocysts and those that arrested before this stage in development (P < 0.01; ANOVA). By examining the chromosome content of each blastomere from cleavage stage embryos via DNA-Seq, we determined that rhesus embryos have an aneuploidy frequency up to ~62% (N = 26) with several embryos exhibiting chromosomal mosaicism between blastomeres (N = 6). Certain blastomeres also exhibited reciprocal whole chromosomal gains or losses, indicating that these embryos had undergone mitotic non-disjunction early in development. In addition, findings of reciprocal sub-chromosomal deletions/duplications among blastomeres suggest that chromosomal breakage had occurred in some embryos as well. Embryo immunostaining for the nuclear envelope protein, LAMIN-B1, demonstrated that fragmented cleavage-stage rhesus embryos often contain micronuclei and that cellular fragments can enclose DNA. Our DNA-Seq analysis confirmed that cellular fragments might encapsulate whole and/or partial chromosomes lost from blastomeres. When embryos were immunostained with gamma-H2AX, a marker of chromatin fragility, we observed distinct foci solely in micronuclei and DNA-containing cellular fragments. This suggests that micronuclei may be ejected from blastomeres through the process of cellular fragmentation and, once sequestered, these mis-segregated chromosomes become highly unstable and undergo DNA degradation. Finally, we also observed that ~10% of embryos prevented cellular fragments or large blastomeres from incorporating into the inner cell mass or trophectoderm at the blastocyst stage (n = 5). Upon confocal imaging, multiple nuclei and intense gamma-H2AX foci were found in a large unincorporated blastomere in one of the blastocysts. Altogether, our findings demonstrate that the rhesus embryo responds to segregation errors by eliminating chromosome-containing micronuclei via cellular fragmentation and/or selecting against aneuploid blastomeres that fail to divide during pre-implantation development with significant implications for human IVF.

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 87-93 ◽  
Author(s):  
Jong Ho Lee ◽  
Joong Hoon Park ◽  
Eun Joo Choi ◽  
Jong Taek Yoon ◽  
Chang Sik Park ◽  
...  
Keyword(s):  
Sex Chromosome ◽  
Chromosome Complement ◽  
Metaphase Plate ◽  
Chromosomal Mosaicism ◽  
Considerable Proportion ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Single Blastomere ◽  
Vinblastine Sulfate

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (>45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.

scholarly journals 331INSEMINATION OF OOCYTES WITH AGED SEMEN ALTERS SEX RATIO OF BOVINE EMBRYOS PRODUCED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 285
Author(s):  
J.L. Edwards ◽  
P. Bredbacka ◽  
A.M. Saxton ◽  
F.N. Schrick
Keyword(s):  
Sex Ratio ◽  
Sperm Motility ◽  
Block Design ◽  
Water Bath ◽  
Bovine Embryos ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Frozen Semen ◽  
Effects Of Aging

Preincubation of semen before insemination may alter the sex ratio of resulting embryos (Lechniak, D. et al., 2003 Reprod. Dom. Anim. 38, 224–227). The overall objective of this study was to evaluate effects of aging sperm before insemination for altering the sex ratio of bovine embryos. In the first experiment oocytes presumed mature were inseminated with frozen semen within minutes post-thaw and percoll-preparation (control) or after aging for 14h post-thaw in a 34.4°C water bath, or after aging for 23h post-thaw at 4°C. Sperm from 4 different bulls, representing 3 different breeds, were utilized (1 bull per experimental replicate). Sperm motility was assessed after aging and percoll preparation. Zona pellucidae were removed from cleaved embryos (43–68h post-insemination; hpi) using 0.5% pronase. Blastomeres were dissociated and counted before transfer to PCR tubes. Sex of cleavage-stage embryos was determined using Ampli-Y™(Bredbacka, P. 1998 Reprod. Nutr. Dev. 38, 605–613). Data were analyzed using a randomized block design with mixed models of SAS (2000) after testing for normality. Aging semen for 14h post-thaw reduced the proportion of motile sperm and compromised the ability of presumptive zygotes to cleave after insemination (Table 1). However, ability of cleaved embryos to develop to the 8–16-cell stage was not affected. Number of blastomeres comprising cleavage-stage embryos that resulted from insemination of oocytes with aged semen was similar to that for controls. Insemination of oocytes with semen aged 14h in a water bath increased the proportion of female embryos (Table 1).To determine if effects of aging semen on altering the sex ratio of bovine embryos was time dependent, oocytes were inseminated with frozen semen within minutes post-thaw (control) or after aging for 8 or 14h post-thaw in a 34.4°C water bath. Sperm from 3 different bulls, representing 2 different breeds were utilized (one bull per experimental replicate). The experiment was replicated 3 times with 195–233 oocytes inseminated within each treatment. Sperm motility averaged 72.7, 65.7 and 45.0% for control and semen aged for 8 and 14h, respectively (SEM=10). Cleavage of inseminated oocytes (67hpi) was similar regardless of sperm treatment (68.5, 70.2 and 70.1%; SEM 14.6, for control semen or after aging for 8 or 14h post-thaw, respectively). Insemination of oocytes with semen aged for 14h tended to increase the proportion of female embryos (51.6 v. 38.3 and 39.1%; sperm aged for 14h v. 8h or control, respectively;; P=0.08; SEM=9.7). Within the seven replicates across both experiments, the differences in percent female for control v. aged were 5.2, 6.3, 14.9, 18.5, 20.0, 29.7 and 60%, with the highest three being significantly different (P&lt;0.05) by Fisher’s Exact test. Bull or replicate variation was noted but the direction of aging for increasing proportion of females was consistent. Preliminary observations suggest that biological differences between X- and Y-bearing sperm may exist such that alternative strategies for altering sex ratio in livestock species may be possible.

scholarly journals Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karina Cañón-Beltrán ◽  
Yulia N. Cajas ◽  
Serafín Peréz-Cerezales ◽  
Claudia L. V. Leal ◽  
Ekaitz Agirregoitia ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Akt Signaling Pathway ◽  
Cell Stage ◽  
Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

O-205 Aneuploidy induces proteotoxic stress and autophagy-mediated apoptosis in human preimplantation embryos

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Regin ◽  
E. Couvreu De Deckersberg ◽  
Y Guns ◽  
P Verdyck ◽  
G Verheyen ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Caspase 3 ◽  
Small Sample ◽  
Confocal Imaging ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Dependent Manner ◽  
Sequencing Data ◽  
Proteotoxic Stress ◽  
Human Preimplantation Embryos

Abstract Study question Are aneuploid cells in human preimplantation embryos eliminated by apoptosis due to proteotoxic stress and autophagy-mediated apoptosis? Summary answer Proteotoxic stress, autophagy and apoptosis are differentially activated in aneuploid embryos, showing that aneuploid cells are eliminated by these mechanisms during early human embryogenesis. What is known already Aneuploidies are a common feature of human preimplantation embryos which could explain low success rates after in vitro fertilization (IVF). While most aneuploidies of meiotic origin are detrimental, transfer of euploid-aneuploid mosaic embryos can lead to healthy live-births. Moreover, the proportion of aneuploid cells are lower in blastocysts when compared to cleavage stage embryos. In the mouse, aneuploid cells are eliminated from the epiblast by autophagy-mediated apoptosis in a p53-dependent manner. We propose that in human embryos, aneuploidy causes chronic protein misfolding which leads to autophagy-induced apoptosis. Study design, size, duration Eighty-one blastocysts that were diagnosed by PGT as euploid (n = 49) or uniformly combined abnormal (CA, n = 32), i.e. 2 or more chromosomes were abnormal in every cell, were warmed. Sixty-seven were suitable for trophectoderm (TE) biopsy, 54 biopsies were successfully tubed and sent for RNA-sequencing while the remainder of the embryos was fixed for immunostaining. Thirty-three day-3 embryos were overnight incubated in 0.5µM reversine allowed to develop into blastocysts and treated as the PGT embryos. Participants/materials, setting, methods After TE biopsy, we live-stained the embryos with either Caspase-3/7 or 8 and subsequently fixed them. The biopsies underwent RNA-sequencing using the SMART-seqv4 and the fixed embryos were immunostained for LC3B, p62 (autophagy) and HSP70 (proteotoxic stress). Confocal imaging was performed using a Zeiss LSM800 confocal microscope and the presence of signal was quantified using the Zen Blue 2.0 and Arivis software. Main results and the role of chance Forty-two percent of the embryos in which we induced aneuploidies using reversine developed into blastocysts, which is comparable to untreated embryos. After immunostaining, we observed that CA and reversine-treated (RT) embryos contained less cells than euploid embryos (median number of nuclei: 43.5, 47, 90, respectively). This correlates with a higher expression of apoptotic markers Caspase-3/7 in CA embryos (p = 0.0199) and Caspase-8 in both aneuploid groups (CA: p = 0.0085 and RT: p = 0.0394). Aneuploid embryos showed significantly increased HSP70 levels (median intensity per cell: euploid=165, CA = 313, RT = 400), LC3B (median puncta per cell: euploid=3.07, CA = 10.10, RT = 19.62) and p62 (median puncta per cell: euploid=17.60, CA = 30.53), suggesting increased proteotoxic stress and autophagy. Preliminary analysis of the RNA-sequencing data reveals enrichment for pathways such as the p53-pathway, protein secretion, TNFA signaling via NFkB and apoptosis, supporting the hypothesis of a link between aneuploidy and apoptosis. Limitations, reasons for caution No functional tests e.g. with inhibitors of autophagy were carried out. RNA-sequencing was carried out on a small sample; we will expand this sample in the near future. Wider implications of the findings This study shows for the first time the mechanism by which aneuploid cells are eliminated from the human preimplantation embryo, explaining how mosaic embryos can still lead to a healthy and genetically normal live birth. Trial registration number not applicable

scholarly journals Effect of Linoleic Acid-Albumin on Post-Thaw Survival of in vitro-Produced Bovine Embryos at the 16-Cell Stage.

2000 ◽  
Vol 62 (4) ◽  
pp. 465-467 ◽  
Author(s):  
eiichiro KTOMINAGA ◽  
Yukako HAMADA ◽  
Tsuyoshi YABUUE ◽  
Tetsushi ARIYOSHI
Keyword(s):  
Linoleic Acid ◽  
Bovine Embryos ◽  
Cell Stage

178 RELATIVE ABUNDANCE OF Oct-4, Nanog, AND Sox-2 TRANSCRIPTS IN PORCINE OOCYTES AND CLEAVAGE-STAGE EMBRYOS PRODUCED VIA FERTILIZATION IN VITRO OR PARTHENOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 168
Author(s):  
L. Magnani ◽  
R. Cabot
Keyword(s):  
Gene Expression ◽  
Similar Pattern ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Developmental Expression ◽  
Fertilization In Vitro ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Parthenogenetic Embryos

Parthenogenetic embryos obtained by electroactivation of mature oocytes have been used as models in developmental studies. The correct gene expression in early cleavage embryos is essential to sustain embryo development. The precise regulation of genes involved in pluripotency (Oct-4, Sox-2, and Nanog) is crucial to the formation of inner cell mass and trophoblast cells. Failure to do so can contribute to impaired development. We hypothesized that porcine embryos produced by fertilization in vitro and parthenogensis would possess a similar pattern of expression of Oct-4, Nanog, and Sox-2 during cleavage development. The objective of this study was to determine the developmental expression pattern of these three transcription factors in porcine oocytes and cleavage-stage embryos produced by either fertilization or parthenogenesis. Messenger RNAwas isolated from pools of 40-150 germinal vesicle (GV)- and MII-arrested oocytes and pools of 2-cell (2c), 4-cell (4c), 8-cell (8c), and blastocyst-stage embryos produced by in vitro fertilization (IVF) or electroactivation. Quantitative real-time PCR was performed following cDNA synthesis. Transcripts for Oct-4, Nanog, Sox-2, andYWHAG (housekeeping gene control) were amplified in duplicate across three to five experimental replicates. Transcripts were quantified using the comparative CT method using YWHAG as internal control and GV stage as normalizing stage. Fold activation and repression were analyzed with ANOVA and Tukey's post-hoc test. Our results show that porcine embryos produced by either IVF or electroactivation possess a similar pattern of pluripotent gene expression during cleavage-stage development. Oct-4 was found to be present in high abundance in the 2-cell parthenogenetic embryos and then repressed at the 8-cell stage (10-fold; P < 0.05, 2c v. 8c). In IVF embryos, Oct-4 was found in significantly higher amount at the 2-cell stage (35-fold; P < 0.05, 2c v. GV). Nanog transcripts were present at low levels from the GV oocyte until the 4-cell stage in both IVF and parthenogenetic embryos and then upregulated 10 000-fold at the 4-cell stage (P < 0.0001, GV v. 4c); at the blastocyst stage, Nanog transcript levels were similar to the levels found in the GV stage oocytes. Sox-2 transcripts were lower in MII oocytes and were significantly upregulated in 8-cell-stage embryos produced by either IVF or electroactivation (9- and 20-fold; P < 0.01, P < 0.0001, MII v. 8c, respectively). In addition, Sox-2 transcripts were significantly higher in parthenogenetic blastocysts compared to IVF-derived blastocysts (P < 0.05). This work demonstrates that cleavage-stage porcine embryos, produced by either electroactivation or IVF, undergo a similar pattern of activation of key regulatory genes; however, the activation method can have an influence on the transcript abundance of specific genes at defined stages.

scholarly journals Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage

Zygote ◽  
1994 ◽  
Vol 2 (4) ◽  
pp. 281-287 ◽  
Author(s):  
Asangla Ao ◽  
Robert P. Erickson ◽  
Robert M.L. Winston ◽  
Alan H Handysude
Keyword(s):  
Mammalian Species ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Cell Stage ◽  
Gonad Differentiation ◽  
Embryonic Genome ◽  
Human Preimplantation Embryos

SummaryGlobal activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

Sign in / Sign up

Export Citation Format

Share Document