scholarly journals Multiplying embryos: experimental monozygotic polyembryony in mammals and its uses

2019 ◽  
Vol 63 (3-4-5) ◽  
pp. 143-155
Author(s):  
Ellen Casser ◽  
Steffen Israel ◽  
Michele Boiani
Keyword(s):  
Embryo Quality ◽  
Interindividual Variation ◽  
Phenotypic Traits ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Species Specific ◽  
Real Gain ◽  
Original Motivation ◽  
Mz Twins ◽  
Regulative Capacity

Monozygotic (MZ) polyembryony is a strategy to increase the output of a single zygote, thereby producing more offspring from a limited number of oocytes. However, MZ twins and multiples (multiplets) of mammals occur rarely in nature, while their generation has been more successful experimentally. In this work, we review some of the methodological, biological and field aspects of experimental MZ polyembryony in mammals. First attempts of mechanical bisection of 2-cell stage rodent embryos provided a proof-of-principle for the survival and independent development of both blastomeres. Subsequently, experiments in other species, particularly sheep and bovine, allowed 2 methods of embryo multiplication to become routine: the separation or biopsy of blastomeres from cleavage-stage embryos and the bisection of morulae and blastocysts. We discuss how the preferable stage of bisection and the success rate can be species-specific. The scope that profited most from experimental MZ polyembryony is the production of additional copies of elite livestock individuals, the reduction of interindividual variation in test groups and the possibility of investigating discordant phenotypic traits in the same genomic background, for instance, comparing an affected twin with its healthy co-twin. By contrast, the original motivation for experimental polyembryony – efficiently generating more offspring out of the same zygote – has not been fulfilled yet. Although embryo splitting leads to an increase in quantity, there is a loss of embryo quality, thus, there is no real gain from artificially generated embryos (yet) in the field of medically assisted reproduction. In conclusion, mammalian zygotes have the regulative capacity to be polyembryonic, but this is not obligate.

Incorporation of Uridine in Cleavage Stage Eggs of the Sea Urchin Paracentrotus Lividus

1971 ◽  
Vol 26 (8) ◽  
pp. 816-821 ◽  
Author(s):  
Larry E. Bockstahler
Keyword(s):  
Ion Exchange ◽  
Sea Urchin ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Paracentrotus Lividus ◽  
Cleavage Stage ◽  
Early Cleavage ◽  
Cell Stage ◽  
Cleavage Stages ◽  
Layer Chromatography

Incorporation of uridine in cleavage stage eggs of the sea urchin Paracentrotus lividus was investigated. It was shown by ion exchange and thin layer chromatography that most of the uridine taken up during the 16-cell stage was converted into UTP with some incorporation into UDP and UMP. Conversion of uridine to these phosphorylated nucleosides occurred throughout early cleavage stages. A very small amount of uridine taken up by cleavage stage eggs is incorporated into RNA heterogeneous in size. This RNA was examined by polyacrylamide gel electrophoresis.

P–080 Higher sperm DNA fragmentation reduces the proportion of good quality embryos at day 5 on IVF and ICSI cycles from unselected males

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
I Hervá. Herrero ◽  
A Pacheco ◽  
R Rivera-Egea ◽  
M Gi. Julia ◽  
A Navarro-Gomezlechon ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Dna Integrity ◽  
Embryo Morphology ◽  
Sperm Dna Fragmentation ◽  
Cleavage Stage ◽  
Sperm Dna ◽  
Embryo Fragmentation ◽  
The Impact

Abstract Study question Does sperm DNA fragmentation (SDF) reduce the ratio of good-quality embryos in day 3 (D3) and day 5 (D5) of embryonic development? Summary answer High sperm DNA fragmentation (SDF >15%) is associated with poor embryo quality at blastocyst-stage per cycle in unselected patients undergoing IVF and ICSI. What is known already It has been shown that the proportion of spermatozoa with DNA fragmentation is higher in infertile men than in semen from fertile men. However, the controversy regarding the impact that sperm genome damage can have on IVF or ICSI treatments is evident in the published literature. The effects of SDF would become evident after activation of the embryonic genome at 8-cell stage, compromising not only the quality of the embryos obtained, but also the reproductive outcomes, as reduced implantation rates, higher miscarriages rates and thus, a decreased chance of pregnancy. Study design, size, duration This multicentric observational retrospective study included 1339 couples who underwent 2759 IVF-ICSI cycles using autologous oocytes from January 2000 to March 2019. All men have an SDF test in their ejaculated spermatozoa by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling). The subjects were divided into two groups according to their sperm DNA integrity: low (≤15%) (n = 2287 cycles) or high (>15%) (n = 472) SDF. Participants/materials, setting, methods Embryo quality was assessed complying morphological standards at cleavage-stage on D3 and at blastocyst-stage on D5 (inner cell mass (ICM) and trophectoderm (TE) grade (A, B, C or D)) in according to ASEBIR’s embryo selection criteria, being embryos of good quality those categorized as A+B. The outcomes were calculated in relation to the total number of zygotes obtained. The results were compared by Student t test; p value <0.05 was considered significant. Main results and the role of chance The SDF average of the low group was 5.8% (95% CI 5.6–5.9) whereas in high group was 23.7% (95% CI 23.0–24.4). The female age was equal, 37.1 years (95%CI 37.0–37.2) and 37.1 years (95% CI 36.8–37.4) respectively. A total of 9796 embryos were evaluated. The optimal cleavage-stage embryo rate per cycle was 25.0% (95% CI 21.7–28.3) (8.0 average cells number, 1.5 embryo fragmentation average, symmetry 1, mononucleated cells) versus 26.7% (95%CI 19.1–34.2) (7.9 average cells number, 1.8 embryo fragmentation average, symmetry 1, mononucleated cells) when comparing between groups (p < 0.001). Blastocyst-stage arrival rate (number of embryos at D5) per cycle was 55.8% (95% CI 54.3–57.2) in ≤ 15% SDF group (embryo quality score was ICM A:12.1%, B:69.5%, C:8.8%, D:4.5%; TE A: 7.5%, B:42.2%, C:42.2%, D:8.1%) and 55.9% (95% CI 52.8–59.1) in the >15% SDF group (ICM A:12.0%, B:68.7%, C:10.6%, D: 5.2%; TE A:9.1%, B:44.8%, C:37.8%, D:8.3%) (p < 0.001). The good quality blastocyst rate per cycle was significantly higher in the group with SDF ≤15%, 27.7% (95%CI 26.5–29.0) versus SDF >15% (27.4% (95%CI 24.6–30.2)). Of the total number of blastocysts, the proportion of A+B blastocyst was 60.5% (95% CI 58.3–62.7) and 64.2% (95% CI 59.2–69.2) (p < 0.001), respectively. Limitations, reasons for caution The retrospective and multicenter nature of this study leads to uncontrolled biases derived from the clinical practice. Although the results were not adjusted for female’s age, it was not statistically different between groups. Embryo morphology evaluation was performed by senior embryologists, it still remains a subjective evaluation, though. Wider implications of the findings: In this study, a higher amount of data was compiled so that a large number of embryos were analyzed. The DNA integrity of the sperm may be an important consideration when poor quality embryos were obtained in the previous cycle when deciding on the next clinical strategy to apply. Trial registration number NA

P–030 A sperm chromatin damage >15% negatively impacts on the quality of embryos obtained from ovum donation ICSI cycles of unselected couples

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Pachec. Castro ◽  
I Hervas ◽  
R Rivera-Egea ◽  
M Gi. Julia ◽  
A Navarro-Gomezlechón ◽  
...  
Keyword(s):  
Clinical Practice ◽  
Dna Fragmentation ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Cleavage Stage ◽  
Ovum Donation ◽  
Embryo Fragmentation

Abstract Study question Is embryo quality downgraded in couples with elevated sperm DNA fragmentation (SDF) in the ejaculated semen of male partner using donated eggs? Summary answer The rate of good quality embryos at day 3 and blastocyst-stage is statistically inferior in males with SDF>15% undergoing ICSI cycles with donated oocytes. What is known already The effect of a damaged paternal chromatin will be shown from the 8-cell stage of embryo development, a time which the genome of the embryo is transcriptionally active. Fertilization with a spermatozoon with fragmented DNA may impair the quality of the embryos obtained per cycle, and therefore reduce the chances of pregnancy. The use of donated oocytes is an ideal model to evaluate the real effect of SDF on embryo quality by standardizing the female factor. In addition, we have a large cohort of ovum donation cases in our history, which allows a more proper evaluation of the effect. Study design, size, duration Retrospective multicentric study including the clinical data of 864 couples of ovum donation program who underwent 1903 ICSI cycles between January 2000 and March 2019. The DNA fragmentation of their ejaculated spermatozoa was measured by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling). Two study groups were created according to the SDF level: ≤15% (low) (n = 1626) or > 15% (high) (n = 277). Participants/materials, setting, methods Embryos were evaluated throughout embryonic development according to classical morphological parameters at day 3 (D3), on cleavage-stage, and at day 5 (D5), on blastocyst-stage (trophectoderm (TE) and inner cell mass (ICM)), following ASEBIR guidelines, categorized from A to D. Embryos scored as A and B were considered to be good quality. The proportion of embryos was calculated according to the total number of correctly fertilized oocytes or zygotes. A p < 0.05 was considered significant. Main results and the role of chance A total of 6130 embryos were evaluated. The SDF average of ≤ 15% group was 5.9% (95%CI 5.7–6.1) and 24.3% (95%CI 23.2–25.3) in the >15% group. The cycle-related characteristics and the seminal parameters were comparable. The proportion of optimal cleavage-stage embryo (number of A+B embryos at D3) per cycle was 21.7% (95%CI 19.0–24.5) (8.1 average cells number, 0.8 embryo fragmentation average, symmetry 1, mononucleated cells) in ≤ 15% SDF group versus 21.1% (95%CI 13.9–28.3) (8.2 cells number average, 1.3 embryo fragmentation average, symmetry 1, mononucleated cells) (p < 0.001). The blastocyst-stage arrival rate (number of embryos at D5) per cycle was higher in the >15% SDF group (p < 0.001), 53.4% (95%CI 48.8–58.1) (TE quality A:20.5%, B:42.5%, C:22.7%, D:14.8%, and the ICM quality A:26.1%, B:52.1%, C:13.2%, D:6.2%) versus 49.9% (95%CI 48.1–51.6) (TE quality A:21.1%, B:42.8%, C:21.85, D:14.1% and ICM A:26.6%, B:55.5%, C:11.1%, D:4.7%) in the low SDF group. The rate of good quality blastocyst (number of quality A+B embryos in D5) per cycle was significantly higher in the couples with low SDF (24.8% (95%CI 23.6–25.9)) than in those with elevated SDF (23.5% (95%CI20.9–26.2)) (p < 0.001). Accordingly, the A+B blastocyst rate divided by the total number of blastocysts was 59.1% (95%CI 56.7–61.4) versus 55.9% (95%CI 49.9–62.0) (p < 0.001), respectively. Limitations, reasons for caution The main limitation is that retrospective design of the study may not eliminate the potential unaccounted-for bias derived from the clinical practice of multiple centers even though both groups were statistically comparable. Also, the assessment of embryo quality is still remaining highly subjective to embryologists. Wider implications of the findings: Although the effect size is small, it may be useful in clinical practice when an ICSI cycle yields no good-quality embryos, as one of the underlying causes of that fact. Knowing the SDF level can be a helpful tool in making subsequent clinical decisions aimed at improving outcomes for couples. Trial registration number Not applicable

Origin and behaviour of pigment cells in sea urchin embryos

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S42-S43 ◽  
Author(s):  
Tetsuya Kominami
Keyword(s):  
Sea Urchin ◽  
Pigment Cell ◽  
Cell Lineage ◽  
Pigment Cells ◽  
Cleavage Stage ◽  
Fate Map ◽  
Blastula Stage ◽  
Cell Stage ◽  
Stage Embryo ◽  
Founder Cells

Sea urchin pluteus larvae contain dozens of pigment cells in their ectoderm. These pigment cells are the descendants of the veg2 blastomeres of the 60-cell stage embryo. According to the fate map made by Ruffins and Ettensohn, the prospective pigment cells occupy the central region of the vegetal plate. Most of these prospective pigment cells exclusively give rise to pigment cells. Therefore, specification of the pigment cell lineage should occur at some point between the 60-cell and mesenchyme blastula stage. However, the detailed process of the specification of the pigment lineage is unknown.When are pigment cells specified? Are cell interactions necessary for the specification? Do founder cells exist? To answer these questions, I treated embryos with Ca2+-free seawater during the cleavage stage and examined the number of pigment cells observed in pluteus larvae. Treatment at 5.5–8.5 h and especially 7.5–10.5 h postfertilisation markedly reduced the number of pigment cells. The decrease was statistically significant. On the other hand, the treatment at 3.5–6.5 h or 9.5–12.5 h never reduced the number of pigment cells. By examining the frequency of the appearance of embryos whose numbers of pigment cells were less than 20, it was also found that the numbers of pigment cells were frequently in multiples of 4. Embryos having 4, 8, 12, 16 and 20 pigment cells were more frequently observed. Statistics indicated that the frequency of appearance was not random. These results indicated that cell contacts are necessary for the specification of pigment cells and that the specification occurs from 7 to 10 h postfertilisation. The results also suggest that the founder cells, if they exist, divide twice before they differentiate into pigment cells.

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.

124. DNA METHYLATION IN THE 2-CELL MOUSE EMBRYO IS THE RESULT OF DNMT ACTIVITY DURING DEVELOPMENT FROM THE ZYGOTE TO THE 2-CELL STAGE

2009 ◽  
Vol 21 (9) ◽  
pp. 43
Author(s):  
Y. Li ◽  
H. D. Morgan ◽  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Culture Conditions ◽  
Early Embryo ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Stage Of Development ◽  
First Time

In an accompanying abstract we show for the first time that global demethylation of both paternally- and maternally-derived genomes occurs prior to syngamy. It is commonly considered that new methylation of the genome does not commence until late in the preimplantation stage. Yet embryos during cleavage stage are known to show DNA methylation. This creates a paradox, if global demethylation occurs by the time of syngamy yet remethylation does not occur until the blastocysts stage, how can cleavage stage embryos possess methylated DNA. We examined this paradox. We examined DNA methylation in 2-cell embryos by confocal microscopy of anti-methylcytosine immunofluorescence and propidium iodide co-staining of whole mounts. We confirmed that DNA in late zygotes was substantially demethylated in both the male and female pronuclei. By the 2-cell stage, embryos collected direct from the oviduct showed high levels of cytosine methylation. We assessed whether this accumulation of cytosine methylation during the early 2-cell stage was a consequence of DNA methyltransferase (DNMT) activity. This was achieved by treating late stage zygotes with the DNMT inhibitor RG108 (5 μM) for the period of development spanning pronuclear stage 5 to early 2-cell stage. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (P<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. Our results show for the first time that de novo methylation of the genome occurs as early as the 2-cell stage of development and that this is mediated by a RG108-sensitive DNMT activity. The results substantially change our understanding of epigenetic reprogramming in the early embryo.

scholarly journals Genome assembly of the tayra (Eira barbara, Mustelidae) and comparative genomic analysis reveal adaptive genetic variation in the subfamily Guloninae

2021 ◽  
Author(s):  
Lorena Derežanin ◽  
Asta Blažytė ◽  
Pavel Dobrynin ◽  
David A. Duchêne ◽  
José H. Grau ◽  
...  
Keyword(s):  
Body Condition ◽  
De Novo ◽  
Species Traits ◽  
Ecological Niches ◽  
Comparative Genomic ◽  
Phenotypic Traits ◽  
Adaptive Genetic Variation ◽  
Eira Barbara ◽  
Species Specific ◽  
Candidate Loci

Species of the mustelid subfamily Guloninae inhabit diverse habitats on multiple continents, and occupy a variety of ecological niches. They differ in feeding ecologies, reproductive strategies and morphological adaptations. To identify candidate loci associated with adaptations to their respective environments, we generated a de novo assembly of the tayra (Eira barbara), the earliest diverging species in the subfamily, and compared this with the genomes available for the wolverine (Gulo gulo) and the sable (Martes zibellina). Our comparative genomic analyses included searching for signs of positive selection, examining changes in gene family sizes, as well as searching for species-specific structural variants (SVs). Among candidate loci that appear to be associated with phenotypic traits, we observed many genes related to diet, body condition and reproduction. For the tayra, which has an atypical gulonine reproductive strategy of aseasonal breeding, we observe species-specific changes in many pregnancy-related genes. For the wolverine, a circumpolar hypercarnivore that must cope with seasonal food scarcity, we observed many specific changes in genes associated with diet and body condition. Despite restricting some of our analyses to single-copy orthologs present in all three study species, we observed many candidate loci that may be linked to species traits related to environment-specific challenges in their respective habitats.

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