45 Embryonic disc development invitro in ovine embryos

2021 ◽  
Vol 33 (2) ◽  
pp. 129
Author(s):  
P. Ramos-Ibeas ◽  
M. Torres-Used ◽  
M. J. Cocero ◽  
P. Marigorta ◽  
R. Alberio ◽  
...  
Keyword(s):  
Culture System ◽  
Cell Number ◽  
Activin A ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Rock Inhibitor ◽  
Embryo Survival ◽  
Embryonic Disc ◽  
Chi Squared ◽  
One Way Anova

Embryonic mortality during the second week of pregnancy has an important economic impact on farming. At this time, the embryo undergoes critical developmental events that cannot be recapitulated invitro, limiting our understanding of these pregnancy losses. After the blastocyst stage, the hypoblast migrates to cover the inner surface of the embryo and the epiblast forms a flat embryonic disc (ED) that initiates gastrulation. The aim of this study was to develop an invitro culture system to support sheep embryo development after the blastocyst stage. Day 6/7 invitro-produced blastocysts were cultured over agarose gels to prevent attachment and allocated to different media: synthetic oviductal fluid with 10% fetal bovine serum (SOF-FBS, n=52), an invitro culture medium (hIVC, n=35) supporting ED formation in human embryos (Deglincerti et al. 2016 Nature 533, 251-254; https://doi.org/10.1038/nature17948), and chemically defined N2B27 medium (n=38) supporting ED formation in bovine embryos (Ramos-Ibeas et al. 2020 Reproduction 160, 579-589, https://doi.org/10.1530/REP-20-0243). At Day 14, survival and embryo area were recorded, the abundance of transcripts encoding interferon Tau (TP1) and metabolic enzymes was analysed by RT-qPCR, and the development of epiblast and hypoblast was assessed by immunostaining for SOX2 and SOX17. Embryo survival and size and the percentage of embryos achieving complete hypoblast migration were significantly reduced in SOF-FBS (Chi-squared test and one-way ANOVA; P<0.05). Only N2B27 medium supported epiblast survival in 11/28 embryos. TP1 expression increased at Day 14 in all culture conditions and metabolism-related genes revealed a shift from anaerobic glycolysis to oxidative phosphorylation after culture in hIVC and N2B27. Next, to promote epiblast development, we allocated blastocysts to N2B27 medium supplemented with activin A (n=45), rho-associated protein kinase (ROCK) inhibitor (ROCKi, n=42), or insulin growth factor 1 (IGF1, n=29). IGF1 reduced significantly the percentage of embryos showing an ED-like structure, whereas activin A supplementation significantly increased epiblast survival (Chi-squared test; P<0.05) and SOX2+ cell number was higher in embryos cultured with ROCK inhibitor. When we combined activin A and ROCKi supplementation (N2B27+A+R, n=151), SOX2+ cell number and the percentage of embryos showing an ED-like structure increased significantly (165.1±53.1 and 31/35 embryos with SOX2+ epiblast cells; ∼89%) compared with N2B27 alone (49.4±12.7 and 5/11; ∼45%) (one-way ANOVA and Chi-squared test; P<0.05). Moreover, 18/31 (∼58%) ED-like structures developed in N2B27+A+R lost the Rauber’s layer (polar trophoblast), and BRACHYURY expression, denoting the onset of gastrulation, was observed in 3/14. In conclusion, we have developed a culture system that supports complete hypoblast migration and ED development invitro, which represents a valuable tool to explore early embryo mortality in livestock species without the need for experimental animals.

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

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
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.

125 EXAMINING CRITERIA FOR EXTENDING BOVINE BLASTOCYST SURVIVAL IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
F. N. T. Cooke ◽  
T. M. Rodina ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Conditioned Medium ◽  
Culture System ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Cell Numbers ◽  
Bovine Blastocyst

Most of the current culture procedures used for bovine in vitro embryo production terminates at the blastocyst stage. Developing procedures for extending embryo lifespan beyond this phase will provide a valuable tool for understanding events that occur during the second week of pregnancy in cattle. The overall objective of the present studies was to identify culture conditions required to support bovine blastocyst development beyond its initial formation. In the first study, individual day 8 blastocysts (day 0 = day of IVF) were cultured until day 11 in 30 µL microdrops of Potassium Simplex Optimized Medium-Bovine Embryo 2 containing 0.1 mm non-essential amino acids or Tissue Culture Medium 199 (M199). Both media were supplemented with 5% [v/v] fetal bovine serum (FBS) and incubations were in an atmosphere of either 5 or 21% (v/v) oxygen. A medium by oxygen interaction (P = 0.007) occurred when assessing cell number on day 11. Blastocysts cultured in M199 and in a 5% O2 environment had greater (P < 0.002) cell numbers (536 � 49) than blastocysts incubated in other conditions (339 � 28). Conditioned medium from blastocysts incubated in 21% O2 contained greater (P < 0.05) concentrations of bioactive interferon-tau (IFNT) than blastocysts incubated in 5% O2 regardless of medium type (70.5 � 28 v. 17.2 � 2.6 ng mL–1). In a follow-up study, blastocysts could remain morphologically viable through day 11 in M199 containing at least 2.5% FBS. To examine whether oxidative stress was responsible for the increase in IFNT production under 21% O2, blastocysts were incubated under a 5% O2 atmosphere in M199 containing 2.5% FBS and increasing concentrations of tert-butylhydroperoxide (tBH), a membrane-permeable oxidative agent. Addition of e3 nm tBH decreased cell numbers but did not increase IFNT concentrations in conditioned medium. To examine whether blastocysts could survive beyond day 11 in culture, day 11 blastocysts were transferred to 400 �L of M199 with 20% FBS under 5% oxygen and cultured from day 11 to 20–21 post-IVF. Half of the medium was replaced every 2–3 days. On day 13–14, 16.6 � 6.1% of blastocysts showed initial signs of degeneration. A portion of blastocysts (32.9 � 9.6%) began attaching to plates on days 13–15 and produced outgrowths that appeared viable on days 20–21. All of the non-attached blastocysts degenerated by day 17–18. No blastocyst elongation was detected. In conclusion, a culture system was developed that sustains blastocyst viability and IFNT production in vitro to day 11. Although this culture system allowed blastocyst survival until day 14, normal conceptus development (i.e. elongation/filamentation) was not achieved. Nonetheless, the culture system provides a useful tool for examining the initial stages of blastocyst development and IFNT production from individual bovine embryos.

scholarly journals Effect of various co-culture systems on embryo development in ovine

2013 ◽  
Vol 58 (No. 10) ◽  
pp. 443-452 ◽  
Author(s):  
B. Heidari ◽  
A. Shirazi ◽  
M.-M. Naderi ◽  
M.-M. Akhondi ◽  
H. Hassanpour ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Cell Number ◽  
Culture Conditions ◽  
The Other ◽  
Culture Period ◽  
Hatching Rate ◽  
Embryonic Fibroblast ◽  
One Way Anova

Considering the advent of mesenchymal stem cells (MSCs) as a new source of somatic cells in embryo co-culture system, the current study was aimed to compare in vitro embryo development using embryonic MSCs monolayer with embryonic fibroblast cells (EFCs), oviductal epithelial cells (OECs), and cell-free culture system. The IVM/IVF presumptive sheep zygotes were randomly cultured in different culture conditions as follows: (1) SOFaaBSA medium for the whole culture period (SOF, n = 371), (2) SOFaaBSA medium for the first 3 days followed by co-culturing with MSCs for the next 5 days (SOF-MSCs, n = 120), (3) co-culturing with MSCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (MSCs-SOF, n = 133), (4) co-culturing with MSCs for the whole culture period (MSCs, n = 212), (5) SOFaaBSA medium for the first 3 days followed by co-culturing with EFCs for the next 5 days (SOF-EFCs, n = 132), (6) co-culturing with EFCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (EFCs-SOF, n = 165), (7) co-culturing with EFCs for the whole culture period (EFCs, n = 236), and (8) co-culturing with OECs for the whole culture period (OECs, n = 255). One-Way ANOVA by multiple pairwise comparisons using Tukey&rsquo;s test was performed. Co-culturing in MSCs group had no superiority over EFCs and OECs groups. Though, when co-culturing with MSCs and EFCs was limited to the first 3 days of culture, the embryo development indices were improved compared to the other co-cultured groups. Considering both the hatching rate and total cell number, the application of MSCs for the first 3 days of culture (MSCs-SOF) was superior to the other co-culture and SOF groups. &nbsp;

scholarly journals The Inclusion Principles of Human Embryos in the WOW-Based Time-Lapse System: A Retrospective Cohort Study

2021 ◽  
Vol 12 ◽  
Author(s):  
Yuqiong Wang ◽  
Sheng Wang ◽  
Xilin Qian ◽  
Yanrong Kuai ◽  
Yang Xu
Keyword(s):  
Culture System ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Individual Identification ◽  
Human Embryos ◽  
Paracrine Signaling ◽  
Negative Effects ◽  
Group Culture ◽  
System A ◽  
Negative Effect

A time-lapse system (TLS) with a well-of-the-well (WOW) dish, which allows individual identification and the possibility of autocrine and paracrine signaling between group-cultured embryos, has been widely used in clinic. However, there is a need to re-think the inclusion principles of human embryos in WOW-based TLS, especially for grade IV (G4) embryos, which are considered to potentially have detrimental effects on surrounding embryos. Here, we carried out a single-center, large-cohort, retrospective study, comprising 303 patients undergoing IVF (148 cases) and ICSI (155 cases), with a total of 3282 embryos, to compare embryonic development until the blastocyst stage in the group culture system with or without G4 embryos. Further, LC-MS/MS was used to analyze the G1-G4 embryo secretome to understand the influence of G4 embryos on the group culture microenvironment. We proved that polypronuclear (PPN) embryos positively contribute to the development of the neighboring embryos through secretion of ILIAP, ITI-H4, and keratin. Existence of more than one G4 embryo had a negative effect on the other embryos (p &lt; 0.05). Moreover, G4 embryos were found to secrete KLKB1 and VTDB, which might harm the neighboring embryos. Thus, our study clarified that when embryos are subjected to group culture in WOW-based TLS, the PPN-derived embryos need not be removed, and it is important to ensure that no more than one G4 embryo is present to avoid negative effects on the neighboring embryos.

scholarly journals Use of Insulin to Increase Epiblast Cell Number: Towards a New Approach for Improving ESC Isolation from Human Embryos

2013 ◽  
Vol 2013 ◽  
pp. 1-7
Author(s):  
Jared M. Campbell ◽  
Michelle Lane ◽  
Ivan Vassiliev ◽  
Mark B. Nottle
Keyword(s):  
Embryonic Stem ◽  
Cell Number ◽  
Poor Quality ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cleavage Stage ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Hesc Derivation

Human embryos donated for embryonic stem cell (ESC) derivation have often been cryopreserved for 5–10 years. As a consequence, many of these embryos have been cultured in media now known to affect embryo viability and the number of ESC progenitor epiblast cells. Historically, these conditions supported only low levels of blastocyst development necessitating their transfer or cryopreservation at the 4–8-cell stage. As such, these embryos are donated at the cleavage stage and require further culture to the blastocyst stage before hESC derivation can be attempted. These are generally of poor quality, and, consequently, the efficiency of hESC derivation is low. Recent work using a mouse model has shown that the culture of embryos from the cleavage stage with insulin to day 6 increases the blastocyst epiblast cell number, which in turn increases the number of pluripotent cells in outgrowths following plating, and results in an increased capacity to give rise to ESCs. These findings suggest that culture with insulin may provide a strategy to improve the efficiency with which hESCs are derived from embryos donated at the cleavage stage.

74 EFFECT OF BETA-MERCAPTOETHANOL ON THE VITRIFICATION CRYOTOLERANCE OF BOVINE IN VITRO-PRODUCED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 137
Author(s):  
E. S. Ribeiro ◽  
M. C. Gonçalves ◽  
M. C. Pedrotti ◽  
L. T. Martins ◽  
R. P. C. Gerger ◽  
...  
Keyword(s):  
Cell Number ◽  
Blastocyst Stage ◽  
High Humidity ◽  
In Vitro Production ◽  
Chi Square ◽  
Embryo Survival ◽  
Chi Square Test ◽  
Vitro Production ◽  
Hatching Rates

The control of oxidative processes in in vitro production (IVP) systems by the use of additives may be an alternative approach to improve embryo cryotolerance. The aim of this study was to verify the effect of β-mercaptoethanol (βME) on the cryotolerance of bovine IVP embryos. In 7 replications, and following IVM-IVF, presumptive zygotes (n = 3735) were in vitro-cultured in SOF medium supplemented or not with 100 μm βME (IVC treatment), at 38.5°C and high humidity. The initial 24 h of IVC was performed in 5% CO2 in air, with the remaining 6 days of IVC carried out in 5% CO2, 5% O2, and 90% N2. On Day 7, resulting blastocysts and expanded blastocysts were vitrified in glass micropipettes in a solution with 20% ethylene glycol + 20% propylene glycol. After warming, embryos were randomly allocated to 1 of 2 sub-groups for an additional 72 h of IVC to the hatching blastocyst (HBL) stage, in fresh SOF medium supplemented or not with 100 μm βME (PVC treatment), at 38.5°C, high humidity and 5% CO2. Experimental groups were as follows: G1 (βME-free medium during IVC and PVC); G2 (βME only during PVC); G3 (βME only during IVC); and G4 (βME during IVC and PVC). Cleavage (Day 2) and blastocyst (Day 7) rates in the IVC treatment and hatching rates (Days 7 to 9) for the PVC treatment were analyzed by the chi-square test, for P < 0.05. Total cell number (TCN) estimated by fluorescence staining in HBL derived from vitrified and nonvitrified embryos was analyzed by ANOVA. The use of βME during IVC did not affect cleavage rates (βME-free, 1491/1858, 80.2% v. βME, 1522/1877, 81.1%), but negatively affected development to the blastocyst stage (βME-free, 813/1858, 43.8% v. βME, 525/1877, 28.0%). Following vitrification, however, βME supplementation during PVC improved hatching rates (G2, 58.1% and G4, 63.8%) compared with groups without the additive (G1, 36.6% and G3, 42.0%). In addition, the presence of βME either during IVC or PVC, or during both culture periods, increased TCN in HBL from vitrified embryos (Table 1). The use of βME during IVC, irrespective of the presence of βME during the PCV period, caused an increase in TCN in HBL in G3 + G4, with no effects on hatching rates (Table 1b), whereas the addition of βME during PVC, irrespective of the presence of βME during the IVC period, resulted in greater hatching rates and TCN in HBL in G2 + G4 than in G1 + G3 (Table 1). In conclusion, the addition of βME during the IVC period did not affect cleavage, but reduced blastocyst yield. Despite that, βME supplementation during the IVC period appeared to have increased the cryotolerance of the resulting blastocysts, expressed by greater TCN in HBL, whereas βME supplementation during the PVC period also improved embryo survival to the vitrification process, manifested by greater hatching rates and TCN in HBL. Table 1.Effect of βME on the cryotolerance of bovine IVP embryos This study was supported by a grant from CNPq/Brazil.

scholarly journals Growth factor expression and function in the human and mouse preimplantation embryo

2002 ◽  
Vol 172 (2) ◽  
pp. 221-236 ◽  
Author(s):  
K Hardy ◽  
S Spanos
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Cell Fate ◽  
Reproductive Tract ◽  
Preimplantation Embryo ◽  
Mammalian Species ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos

There is increasing evidence that even before implantation, human development is regulated by embryonically and maternally derived growth factors. Studies in other mammalian species have shown that growth factors and their receptors are expressed by the preimplantation embryo and the reproductive tract. Furthermore, a number of growth factors have been shown to affect rate of embryo development, the proportion of embryos developing to the blastocyst stage, blastocyst cell number, metabolism and apoptosis. Growth factor ligands and receptors are also expressed in human embryos and the maternal reproductive tract, and supplementation of culture medium with exogenous growth factors affects cell fate, development and metabolism of human embryos in vitro. Autocrine, paracrine and endocrine pathways that may operate within the embryo and between the embryo and the reproductive tract before implantation are proposed.

scholarly journals Alterations of miR-16, miR-let-7a and their target genes expression in human blastocysts following vitrification and re-vitrification

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Maryam Daneshvar ◽  
Mansoureh Movahedin ◽  
Mohammad Salehi ◽  
Mehrdad Noruzinia
Keyword(s):  
Target Genes ◽  
Reproductive Technology ◽  
Blastocyst Stage ◽  
Embryo Cryopreservation ◽  
Human Embryos ◽  
Embryo Survival ◽  
Apoptotic Gene ◽  
Genes Expression ◽  
Family Balancing

AbstractEmbryo cryopreservation is a widely used technique in infertility management and today is an essential part of assisted reproductive technology (ART). In some cases, re-vitrification can be applied to good quality supernumerary warmed embryos that have not been transferred in the present cycle. However, there is no study about re-vitrification impact on microRNA and gene expression in human embryos. The purpose of this study is to evaluate miR-16, miR-let7a and target genes expression in in vitro produced human blastocysts following re-vitrification.Day3 embryos obtained from ICSI cycles of fertile couples referring for family balancing program were biopsied and cultured individually. On the fourth day (post-ICSI) male ones (choices of their parents) were transferred and the females (good quality embryos) were donated for research. Donated embryos were cultured to blastocyst stage and assigned to three groups: fresh, vitrified and re-vitrification. Embryos were vitrified on Cryotech carriers. Then blastocysts of three groups were individually assessed for expression of miR-16, miR-let7a and target genes.The results showed that re-vitrification of human blastocysts did not affect the ability to re-expand in culture. In addition, significant decrease was observed in miR-16 and miR-let7a expression in re-vitrified group compared to fresh (p < 0.05). A significant upregulation of the target genes ITGβ3 and BCL-2 in re-vitrified and vitrified embryos was observed compared to the fresh group (p < 0.05). The expression of BAX as a pro-apoptotic gene showed a significant decrease in re-vitrification group comparing with the fresh one (P < 0.05).The results of this research indicated that re-vitrification of embryos changes the expression of miR-16, miR-let-7a and their target genes. These alterations include increased expression of BCl-2 and ITGβ3 genes which play important roles in embryo survival and implantation, respectively. Clinical proof of these effects requires further research.

Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences

2021 ◽  
Author(s):  
P Stamatiadis ◽  
A Boel ◽  
G Cosemans ◽  
M Popovic ◽  
B Bekaert ◽  
...  
Keyword(s):  
Blastocyst Stage ◽  
S Phase ◽  
Preimplantation Development ◽  
Human Embryos ◽  
Immunofluorescence Analysis ◽  
Media Control ◽  
Cas9 Protein ◽  
M Phase ◽  
Developmental Capacity

Abstract STUDY QUESTION What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model? SUMMARY ANSWER POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation. WHAT IS KNOWN ALREADY Clustered regularly interspaced short palindromic repeats—CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage. STUDY DESIGN, SIZE, DURATION The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain—B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts. LIMITATIONS, REASONS FOR CAUTION One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis. STUDY FUNDING/COMPETING INTEREST(S) The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

scholarly journals Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Reproduction ◽  
2006 ◽  
Vol 131 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Mark G Larman ◽  
Courtney B Sheehan ◽  
David K Gardner
Keyword(s):  
Ethylene Glycol ◽  
Zona Pellucida ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Initial Increase ◽  
Cell Stage ◽  
Free Treatment ◽  
Zona Hardening ◽  
Free Media ◽  
Oocyte Freezing

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.

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