scholarly journals Embryonic lethality in mice lacking Trim59 due to impaired gastrulation development

2017 ◽  
Author(s):  
Xiaomin Su ◽  
Chenglei Wu ◽  
Xiaoying Ye ◽  
Ming Zeng ◽  
Zhujun Zhang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Actin Polymerization ◽  
Critical Role ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Germ Layer ◽  
Layer Formation ◽  
Early Embryo Development ◽  
Germ Layer Formation

ABSTRACTTRIM family members have been implicated in a variety of biological processes such as differentiation and development. We here found that Trim59 plays a critical role in early embryo development from blastocyst stage to gastrula. There existed delayed development and empty yolk sacs from embryonic day (E) 8.5 inTrim59-/- embryos. No viableTrim59-/- embryos were observed beyond E9.5. Trim59 deficiency affected primary germ layer formation at the beginning of gastrulation. InTrim59-/- embryos at E6.5 and E7.5, the expression of primary germ layer formation associated genes includingBrachyury,lefty2,Cer1,Otx2,Wnt3andBMP4was reduced. Homozygous mutant embryonic epiblast was contracted and the mesoderm was absent. Trim59 could interact with actin and myosin associated proteins. Trim59 deficiency disturbed F-actin polymerization during inner cell mass differentiation. Trim59 mediated polymerization of F-actin was via WASH K63-linked ubiquitination. Thus, Trim59 may be a critical regulator for early embryo development from blastocyst stage to gastrula through modulating F-actin assembly.

213 HOXB9 PROTEIN IS PRESENT THROUGHOUT EARLY EMBRYO DEVELOPMENT IN THE BOVINE

2013 ◽  
Vol 25 (1) ◽  
pp. 255
Author(s):  
C. Sauvegarde ◽  
D. Paul ◽  
R. Rezsohazy ◽  
I. Donnay
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mature Oocyte ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Immature Oocyte ◽  
Cell Stage ◽  
Morula Stage ◽  
Oocyte Stage

Hox genes encode for homeodomain transcription factors well known to be involved in developmental control after gastrulation. However, the expression of some of these genes has been detected during oocyte maturation and early embryo development. An interesting expression profile has been obtained for HOXB9 in the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436): its relative expression increases between the immature oocyte and the zygote, further increases at the 5- to 8-cell stage to peak at the morula stage before decreasing at the blastocyst stage. The main objective of this work is to establish the HOXB9 protein profile from the immature oocyte to the blastocyst in the bovine. Bovine embryos were produced in vitro from immature oocytes obtained from slaughterhouse ovaries. Embryos were collected at the following stages: immature oocyte, mature oocyte, zygote (18 h post-insemination, hpi), 2-cell (26 hpi), 5 to 8 cell (48 hpi), 9 to 16 cell (96 hpi), morula (120 hpi), and blastocyst (180 hpi). The presence and distribution of HOXB9 proteins were detected by whole-mount immunofluorescence followed by confocal microscopy using an anti-human HOXB9 polyclonal antibody directed against a sequence showing 100% homology with the bovine protein. Its specificity to the bovine protein was controlled by Western blot on total protein extract from the bovine uterus and revealed, among a few bands of weak intensities, 2 bands of high intensity corresponding to the expected size. Oocytes or embryos were fixed and incubated overnight with rabbit anti-HOXB9 (Sigma, St. Louis, MO, USA) and mouse anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA) primary antibodies and then for 1 h with goat anti-rabbit Alexafluor 555 conjugated (Cell Signaling Technology, Beverly, MA, USA) and goat anti-mouse FITC-conjugated (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibodies. Embryos were then mounted in Vectashield containing DAPI. HOXB9 is detected from the immature oocyte to the blastocyst stage. At the immature oocyte stage, it is mainly localised in the germinal vesicle with a weak signal in the cytoplasm. At the mature oocyte stage, HOXB9 labelling is present in the cytoplasm. At the zygote stage, a stronger immunoreactivity is observed in the pronuclei than in the cytoplasm. From the 2-cell stage to the morula stage, the presence of HOXB9 is also more important in the nuclei than in the cytoplasm. HOXB9 is also observed at the blastocyst stage where it is localised in the nuclei of the trophectoderm cells, whereas an inconstant or weaker labelling is observed in the inner cell mass cells. In conclusion, we have shown for the first time the presence of the HOXB9 protein throughout early bovine embryo development. The results obtained suggest the presence of the maternal HOXB9 protein because it is already detected before the maternal to embryonic transition that occurs during the fourth cell cycle in the bovine. Finally, the pattern obtained at the blastocyst stage suggests a differential role of HOXB9 in the inner cell mass and trophectoderm cells. C. Sauvegarde holds a FRIA PhD grant from the Fonds National de la Recherche Scientifique (Belgium).

200 EFFECT OF EXOGENOUS PROGESTERONE DURING IN VITRO CULTURE ON EARLY EMBRYO DEVELOPMENT IN CATTLE

2008 ◽  
Vol 20 (1) ◽  
pp. 179
Author(s):  
M. Clemente ◽  
P. Lonergan ◽  
C. Borque ◽  
J. de La Fuente ◽  
D. Rizos
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Embryo Development ◽  
Mineral Oil ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Early Embryo Development ◽  
Significant Difference ◽  
The Media

Preimplatation embryos grown in vitro are sensitive to their environment, and the conditions of culture can affect developmental potential. Progesterone (P4) is the key hormone responsible for maintenance of pregnancy in mammals, and circulating levels in the early postconception period have been associated with pregnancy success. It is not clear whether P4 acts directly or indirectly on the embryo to alter gene expression and development. The aim of this study was to assess the effect of varying levels of exogenous P4 on the development of bovine zygotes to the blastocyst stage in vitro. A preliminary study was conducted to analyze the media used for culture (stock of P4, SOF, SOF + 1 × 10–7 M P4) on Days 1 (day of culture), 4, and 7 for P4 concentration in 25-μL droplets overlain with mineral oil or 500 μL in wells with or without mineral oil. P4 was measured using an ELISA kit, prepared for human serum or plasma (DE1561 Dimeditec Diagnostics GmbH, Kiel, Germany). Inter- and intra-assay coefficients of variation were 6.63 and 6.42%, respectively, and recovery was 95%. P4 concentration on Day 1 in all media was the expected (40 ng mL–1). However, on Days 4 and 7 in media under mineral oil, the level of P4 was nearly zero (0.1 to 1.6 ng mL–1) compared with the media without mineral oil, which remained unchanged (39 to 40 ng mL–1) through the 7 days of culture. Zygotes (n = 1467) were produced in 8 replicates by in vitro oocyte maturation and fertilization, and were cultured in groups of 40 to 50 in wells of 500 μL without mineral oil in (1) SOF supplemented with 5% fetal calf serum (control–), (2) SOF with ethanol (control+), (3) SOF with P4 0.1 × 10–7 M, (4) SOF with P4 1 × 10–7 M, and (5) SOF with P4 10 × 10–7 M at 39°C, 5% CO2 and 5% O2, with maximum humidity. No significant difference was found between groups in cleavage rate or blastocyst yield on Days 6, 7, and 8 (Table 1). These results indicate that the addition of P4 to the in vitro culture medium (SOF) did not enhance the development of bovine embryos to the blastocyst stage. However, further studies on the quality of these embryos in terms of gene expression are in preparation. Table 1. Effect of P4 on bovine in vitro early embryo development

86 EFFECT OF LACTATION ON EMBRYO DEVELOPMENT DURING THE POSTPARTUM PERIOD IN DAIRY COWS

2012 ◽  
Vol 24 (1) ◽  
pp. 155 ◽  
Author(s):  
V. Maillo ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Garrett ◽  
A. K. Kelly ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Embryo Development ◽  
Metabolic Profile ◽  
Reproductive Tract ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Blood Samples ◽  
Early Embryo Development ◽  
Lactating Cows

The aim of this study was to examine the effect of lactation and associated metabolic profiles on the ability of the reproductive tract of postpartum dairy cows to support early embryo development. Twenty-one age-matched primiparous Holstein cows were used. Immediately after calving, half of the cows were dried off while the remainder were milked twice daily. To characterise the metabolic profile of the cows, jugular blood samples were taken twice weekly starting 15 days before calving until Day 100 postpartum. At the same time, bodyweight (BW) and body condition score (BCS) were recorded. In Experiment 1, around Day 60 postpartum, the oestrous cycles of all cows were synchronized and sixty-five 2- to 4-cell in vitro-produced embryos were endoscopically transferred on Day 2 (Day 0 = oestrus) to the oviduct ipsilateral to the corpus luteum. On Day 7, the oviduct and uterus were flushed endoscopically and the number of embryos developing to the blastocyst stage was recorded. In Experiment 2, around Day 95 postpartum, cows were re-synchronized and 15 to 20 in vitro-produced blastocysts were transferred to the uterine horn ipsilateral to the corpus luteum. On Day 14, conceptuses were recovered by flushing the reproductive tract at slaughter and were measured. Jugular blood samples were taken daily from Day 0 to 7 (Exp. 1) or 14 (Exp. 2) to measure serum concentrations of progesterone. Data were analysed by ANOVA. Concentrations of NEFA and β-HB were higher (P ≤ 0.05) and glucose, insulin and IGF-1 were lower (P ≤ 0.05) in lactating compared with dry cows. BW and BCS were significantly higher in the non-lactating cows throughout the postpartum period. Recovery rates in both experiments were similar between groups (Exp. 1: 63.9 ± 7.2 vs 65.6 ± 8.6 and Exp 2: 33.3 ± 9.6 vs 39.8 ± 9.6 for dry and milking cows, respectively). In Exp. 1, of the structures recovered, significantly more developed to the blastocyst stage in the dry than in lactating cows (49.3 ± 3.8 vs 32.6.3 ± 4.4, respectively; P ≤ 0.05). Progesterone concentrations did not differ between groups. In Exp. 2, no differences were observed in terms of conceptus dimensions on Day 14 (n = 152). Progesterone concentrations were higher in lactating cows from Day 9 to 14 (P ≤ 0.05). In conclusion, this study provides evidence that at 60 days postpartum, the reproductive tract of lactating cows is compromised in its ability to support early embryo development compared with age-matched parous non-lactating cows; however, by 95 days postpartum there was no apparent difference in conceptus development, consistent with less metabolic stress as indicated by the metabolic profile. Funded by Science Foundation Ireland (SFI/07/SRC/B1156) and the Spanish Ministry of Science and Innovation (AGL2009-11810). VM was supported by an STSM award from the COST Action FAO7O2.

63 Effect of sire conception rate on bovine early embryo development

2021 ◽  
Vol 33 (2) ◽  
pp. 138
Author(s):  
K. Clark ◽  
J. N. Drum ◽  
J. A. Rizo ◽  
M. S. Ortega
Keyword(s):  
Embryo Development ◽  
Gradient Centrifugation ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Conception Rate ◽  
Cell Stage ◽  
Fluorescent Intensity ◽  
Early Embryo Development ◽  
High Performing ◽  
Sire Conception Rate

Currently, the only measure of sire fertility in the bovine is sire conception rate (SCR), which is determined by Day 70 pregnancy diagnosis and not reflective of early embryo development. Therefore, this study aimed to establish the relationship between SCR and early embryo development. In the first experiment, 65 sires of negative (<−1, n=25), average (−1 to 1, n=19), and high (> +1, n=21) SCR were characterised for their ability to produce embryos using an invitro embryo production (IVP) system. For each sire, 100 cumulus–oocyte complexes (COCs) were used. COCs were matured for 22h, fertilized by co-incubation with sperm selected from density gradient centrifugation for 18h, and then placed in culture medium. A sire of known IVP performance was used as a control in each run. Cleavage and blastocyst rates (BL) were measured on Days 3 and 8 post-insemination, respectively. Photographs were taken on Days 3, 5, and 8 to identify arrest stages of non-blastocyst embryos. Sires were ranked based on their blastocyst rate and grouped into quartiles for statistical analysis. Differences in BL were determined by ANOVA using sire, IVP run, and a sire×IVP run interaction. In addition, the correlation between SCR and BL was determined. All data were analysed using SAS software version 9.4 (SAS Institute Inc.). Mean BL between each quartile was significant (P<0.05), with rates ranging from 8 to 22% and 32 to 62% for the lowest and highest quartile, respectively. There was no correlation (P=0.90) between SCR and BL. Arrest stage was measured by subtracting the number of Day-8 blastocysts from, first, embryos that were morulas on Day 5, and then embryos that were 8- to 16-cell stage embryos on Day 5. This method is based on the assumption that embryos closer to the blastocyst stage on Day 5 are more likely to contribute to the Day 8 blastocyst population. The most frequent arrest stage was the 4- to 6-cell stage (39/52 sires). It has been shown that decreased rates of autophagy are associated with embryonic arrest at the 4- to 8-cell stage in humans, leading us to investigate this mechanism in the second experiment. Select high (n=3) and low (n=4) performing sires identified in experiment 1 were used to generate 4- to 6-cell embryos, and autophagy rates were measured using live immunofluorescence with CYTO-ID autophagy dye (n=20 embryos/sire). The mean fluorescent intensity of each embryo was divided by the number of cells within the embryo. Differences in autophagy between high and low sires were determined by ANOVA using SAS. Interestingly, low-performing sires had a significantly higher autophagy rates than high-performing sires (77.8±3.1 vs. 50.0±3.5). This could indicate that embryos produced with low-performing sires had higher levels of stress than their counterparts. In summary, the effect of sire on embryonic development seems to be independent of the SCR classification. The most common arrest stage observed is the 4- to 6-cell stage, right before embryonic genome activation. Further research is required to elucidate the mechanisms by which sires influence pre-implantation development. This research was supported by USDA-NIFA AFRI Competitive Grant No. 2019-67015-28998.

scholarly journals Inhibition of DNA repair protein RAD51 affects porcine preimplantation embryo development

Reproduction ◽  
2019 ◽  
Vol 157 (3) ◽  
pp. 223-234 ◽  
Author(s):  
Zhe-Long Jin ◽  
Xing-Hui Shen ◽  
Liang Shuang ◽  
Jeong-woo Kwon ◽  
Min-Jeong Seong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Embryo Development ◽  
Critical Role ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Preimplantation Embryo Development ◽  
Γh2ax Foci ◽  
Repair Protein ◽  
Dna Repair Protein

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)–p53–p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.

scholarly journals Does miRNA Expression in the Spent Media Change During Early Embryo Development?

2021 ◽  
Vol 8 ◽  
Author(s):  
Paul Del Rio ◽  
Pavneesh Madan
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Rna Extraction ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Early Embryo Development ◽  
Total Rna ◽  
Implantation Period ◽  
Spent Media

Distinct miRNA populations have been detected in the spent media of in-vitro culture systems. However, profiling has been limited to media conditioned with blastocyst-stage embryos. Therefore, the aim of the study was to profile extracellular miRNAs throughout the pre-implantation period in bovine embryos. To achieve this, cumulus oocyte complexes were aspirated from ovaries, in-vitro matured, fertilized, and cultured under standard laboratory procedures to the 2-cell, 8-cell, or blastocyst stage of development. At each developmental stage, 25 μl of spent in-vitro culture media was collected, pooled to 300 μl, and processed for total RNA extraction. In-vitro culture media, which never came in contact with any embryos, were additionally processed for total RNA extraction to serve as a negative control. Following hybridization on a GeneChip miRNA 4.0 array, comparative analysis was conducted between spent media and control samples. In total, 111 miRNAs were detected in the spent media samples, with 56 miRNAs identified in blastocyst spent media, 14 miRNAs shared between 8-cell and blastocyst spent media, 7 miRNAs shared between all 3 conditions, and 6 miRNAs exclusive to 2-cell spent media. miRNA-mRNA target prediction analysis revealed that the majority of genes predicted to be regulated by the miRNAs identified in the study have roles in cellular process, metabolic process, and biological regulation. Overall, the study suggest that miRNAs can be detected in the spent media of in-vitro culture system throughout the pre-implantation period and the detected miRNAs may influence genes impacting early embryo development.

182 EFFECTS OF MITOCHONDRIAL ACTIVITY OF INJECTED SPERM ON EARLY DEVELOPMENT IN BOVINE INTRACYTOPLASMIC SPERM INJECTION-DERIVED EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 205
Author(s):  
Y. Nagao ◽  
H. Yamamoto ◽  
B. Sarentonglaga ◽  
K. Ogata ◽  
M. Yamaguchi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Laser Scanning ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mitochondrial Activity ◽  
Early Embryo Development

Intracytoplasmic sperm injection (ICSI) has become the method of choice for bovine ovum pick-up and IVF. However, there are many difficulties with the ICSI technique to obtain viable fetuses. One of the major problems associated with this technique is our lack of knowledge concerning the status of the sperm mitochondria when injected into the oocyte and its effect on embryo development. First, we examined the mitochondrial activity of sperm that had been activated by culturing with methyl-β cyclodextrin (MBCD), in ICSI and in IVF. In vitro-matured oocytes and JC1-labelled sperm were used for the ICSI and IVF. The fluorescence intensity of injected/penetrated sperm mitochondria was measured using confocal laser scanning microscopy. Then, the relative membrane potential of the mitochondria was analysed by a ratiometric method. Second, the reactive oxygen species (ROS) production and capacitation status of the sperm exhibiting normal motility and of the sperm that had been activated by culturing with MBCD were analysed. The ROS levels produced by the sperm were estimated using the luminol assay. The chlortetracycline stain was used to evaluate capacitation status of the sperm. Third, the effect of ROS produced by these sperm types upon embryogenesis following ICSI and IVF was studied. Early developing embryos were examined with a stereomicroscope for cleavage and development to the blastocyst stage after 7 days of culture. Chromosome samples stained with Giemsa solution from the blastocysts were used to analyse the chromosomal integrity. Data were analysed by t-test for Experiments 1 and 2, and ANOVA with Fisher's PLSD test for Experiment 3. The mitochondrial activity immediately after ICSI was higher than at 3 h after insemination (immediately after sperm penetration) in IVF (P < 0.05). The sperm exhibiting activation were capacitated and produced more ROS than the sperm exhibiting normal motility (P < 0.05). The rates of cleaved embryo and blastocyst after ICSI with activated sperm were the same as that in ICSI with normal motility sperm and in IVF (cleaved rate: 66.7, 71.8, and 85.0%, respectively; blastocyst rate: 24.4, 23.3, and 32.0%, respectively). However, chromosomal integrity of blastocysts derived from ICSI with activated sperm was lower than that for ICSI with normal motility sperm or for IVF (23.1, 75.0, and 63.6%, respectively; P < 0.01). In conclusion, capacitated, activated sperm induced chromosomal aberrations during early embryo development following ICSI. Conceivably, the selection of sperm exhibiting progressive motility, which is expected to be activated and to fertilize, would not always be better for early embryo development and fetal growth following ICSI due to the ROS derived from the sperm mitochondria. Injection of sperm exhibiting normal motility, or of mitochondria reduced activated sperm, could improve the quality of ICSI-derived embryos.

scholarly journals Comprehensive Transcriptome Analysis of mRNA Expression Patterns of Early Embryo Development in Goat under Hypoxic and Normoxic Conditions

Biology ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 381
Author(s):  
Yongjie Wan ◽  
Dongxu Li ◽  
Mingtian Deng ◽  
Zifei Liu ◽  
Liang Liu ◽  
...  
Keyword(s):  
Embryo Development ◽  
Expression Patterns ◽  
Interaction Network ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Functional Enrichment ◽  
Gene Interaction Network ◽  
Cell Stage ◽  
Early Embryo Development ◽  
Pathway Gene

It has been reported that hypoxic environments were more suitable for the in vitro development of mammalian embryos, but the underlying mechanisms were still unclear. In the present study, RNA-seq was performed to compare 8-cell-stage and blastocyst-stage goat embryos under hypoxic and normoxic conditions; zygotes were checked at 72 and 168 h to 8-cell stage (L8C) and blastocyst stage (LM) in hypoxic conditions and 8-cell stage (H8C) and blastocyst stage (HM) in normoxic conditions. In the H8C and L8C groups, 399 DEGs were identified, including 348 up- and 51 down-regulated DEGs. In the HM and LM groups, 1710 DEGs were identified, including 1516 up- and 194 down-regulated DEGs. The expression levels of zygotic genes, transcription factors, and maternal genes, such as WEE2, GDF9, HSP70.1, BTG4, and UBE2S showed significant changes. Functional enrichment analysis indicated that these DEGs were mainly related to biological processes and function regulation. In addition, combined with the pathway–gene interaction network and protein–protein interaction network, twenty-two of the hub genes were identified and they are mainly involved in energy metabolism, immune stress response, cell cycle, receptor binding, and signal transduction pathways. The present study provides comprehensive insights into the effects of oxidative stress on early embryo development in goats.

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