scholarly journals Embryo aggregation regulates in vitro stress conditions to promote developmental competence in pigs

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e8143 ◽  
Author(s):  
Pil-Soo Jeong ◽  
Seung-Bin Yoon ◽  
Mun-Hyeong Lee ◽  
Hee-Chang Son ◽  
Hwal-Yong Lee ◽  
...  
Keyword(s):  
Large Scale ◽  
Formation Rate ◽  
Underlying Mechanism ◽  
Stress Conditions ◽  
Developmental Competence ◽  
Scale Production ◽  
Cell Stage ◽  
Beneficial Effects ◽  
The Relationship

Embryo aggregation is a useful method to produce blastocysts with high developmental competence to generate more offspring in various mammals, but the underlying mechanism(s) regarding the beneficial effects are largely unknown. In this study, we investigated the effects of embryo aggregation using 4-cell stage embryos in in vitro developmental competence and the relationship of stress conditions in porcine early embryogenesis. We conducted aggregation using the well of the well system and confirmed that aggregation using two or three embryos was useful for obtaining blastocysts. Aggregated embryos significantly improved developmental competence, including blastocyst formation rate, blastomere number, ICM/TE ratio, and cellular survival rate, compared to non-aggregated embryos. Investigation into the relationship between embryo aggregation and stress conditions revealed that mitochondrial function increased, and oxidative and endoplasmic reticulum (ER)-stress decreased compared to 1X (non-aggregated embryos) blastocysts. In addition, 3X (three-embryo aggregated) blastocysts increased the expression of pluripotency, anti-apoptosis, and implantation related genes, and decreased expression of pro-apoptosis related genes. Therefore, these findings indicate that embryo aggregation regulates in vitro stress conditions to increase developmental competence and contributes to the in vitro production of high-quality embryos and the large-scale production of transgenic and chimeric pigs.

scholarly journals Effects of Escherichia coli- and Staphylococcus aureus-induced mastitis in lactating cows on oocyte developmental competence

Reproduction ◽  
2014 ◽  
Vol 147 (1) ◽  
pp. 33-43 ◽  
Author(s):  
S Asaf ◽  
G Leitner ◽  
O Furman ◽  
Y Lavon ◽  
D Kalo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Transvaginal Ultrasound ◽  
Formation Rate ◽  
Developmental Competence ◽  
Cortical Granule ◽  
Disruptive Effect ◽  
Cell Stage ◽  
Oocyte Developmental Competence

Mastitis is associated with decreased fertility in dairy cows. In the current study, we created an experimental model to simulate short-term mastitis by a single intramammary administration of Gram-negative endotoxin ofEscherichia coliorigin (G−), or Gram-positive toxin ofStaphylococcus aureusorigin (G+), to examine the effect of mastitis on oocyte developmental competence. Healthy Holstein cows were synchronized, and follicular fluid (FF) of cows treated with G+ or G− and of uninfected cows (controls) was aspirated from the preovulatory follicles by transvaginal ultrasound procedure. The aspirated FF was used as maturation medium forin vitroembryo production. The distribution of matured oocytes into different cortical granule classes and meiotic stages was affected by G− administration (P<0.05) but not by G+ administration. The proportion of oocytes that cleaved to two- and four-cell stage embryos (44 h postfertilization) was lower in both G+ and G− groups than in controls (P<0.05). Blastocyst formation rate (7–8 days postfertilization) was lower in the G− group (P<0.05) and numerically lower in the G+ group compared with their uninfected counterparts. The total cell number in blastocysts did not differ among groups; however, the apoptotic index was higher in the G+ group (P<0.05), but not in the G− group, relative to controls. Examining mRNA relative abundance in oocytes and early embryos revealed mastitis-induced alterations inPTGS2(COX2),POU5F1, andHSF1but not inSLC2A1(GLUT1) orGDF9. Results indicate a differential disruptive effect of mastitis induced by G− and G+ on oocyte developmental competence in association with alterations in maternal gene expression.

Sodium butyrate improves the cloned yak embryo viability and corrects gene expression patterns

Zygote ◽  
2013 ◽  
Vol 23 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Xian-rong Xiong ◽  
Dao-liang Lan ◽  
Jian Li ◽  
Yong Wang ◽  
Jin-cheng Zhong
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Formation Rate ◽  
Cell Number ◽  
Developmental Competence ◽  
Gene Expression Patterns ◽  
Embryo Viability ◽  
Cell Stage ◽  
Expression Levels

SummaryInterspecies somatic cell nuclear transfer (iSCNT), a powerful tool in basic scientific research, has been used widely to increase and preserve the population of endangered species. Yak (Bos grunniens) is one of these species. Development to term of interspecies cloned yak embryos has not been achieved, possibly due to abnormal epigenetic reprogramming. Previous studies have demonstrated that treatment of intraspecies cloned embryos with (NaBu) significantly improves nuclear–cytoplasmic reprogramming and viability in vitro. Therefore, in this study, we evaluated the effect of optimal NaBu concentration and exposure time on preimplantation development of yak iSCNT embryos and on the expression patterns of developmentally important genes. The results showed that 8-cell rate, blastocyst formation rate and total cell number increased significantly compared with their untreated counterparts when yak iSCNT embryos were treated with 5 nM NaBu for 12 h after activation, but that the 2-cell stage embryo rate was not significantly different. The treatment of NaBu also increased significantly the expression levels of Oct-4 and decreased the expression levels of HDAC-2, Dnmt-1 and IGF-1; the expression patterns of these genes were more similar to that of their bovine–yak in vitro fertilization (BY-IVF) counterparts. The results described above indicated that NaBu treatment improved developmental competence in vitro and ‘corrected’ the gene expression patterns of yak iSCNT embryos.

295 EFFECT OF TREHALOSE DURING IN VITRO MATURATION OF PIG OOCYTES ON OOCYTE MATURATION AND EMBRYONIC DEVELOPMENT AFTER PARTHENOGENESIS

2015 ◽  
Vol 27 (1) ◽  
pp. 236
Author(s):  
Y. Jeon ◽  
B. Baasanjav ◽  
Y. I. Jeong ◽  
Y. W. Jeong ◽  
Y. W. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Large Scale ◽  
Treated Group ◽  
Developmental Competence ◽  
Control Group ◽  
Scale Production ◽  
Developmental Potential

Autophagy is a critical process for the maintenance of cellular homeostasis and mammalian early embryogenesis. Autophagy can be regulated by various chemical inducers. However, there are few reports about effect of autophagy inducer in vitro maturation (IVM) of porcine oocyte. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Immature oocytes were treated with various concentrations (0, 25, 50, and 100 mM) of trehalose in M-199 (Invitrogen, Carlsbad, CA) supplemented with 10 ng mL–1 of epidermal growth factor (EGF; Sigma-Aldrich Corp.), 1 ug mL–1 of insulin (Sigma-Aldrich Corp.), 4 IU mL–1 of pregnant mare serum gonadotropin (PMSG; Intervet, Boxmeer, Holland), 4 IU mL–1 of human chorionic gonadotropin (hCG; Intervet), and 10% (vol/vol) porcine follicular fluid (pFF) for 10 h, and transferred to another IVM medium without trehalose. Osmolality of each groups (0, 25, 50, and 100 mM trehalose) was in the 290 to 295, 310 to 315, 330 to 335, and 375 to 380 osmol range, respectively. After 44 h of IVM, trehalose treatment during IVM did not improve nuclear maturation rates of oocytes in any group (90.7, 92.1, 92.7, and 90.1%, respectively). The developmental competence of oocytes matured with different trehalose concentrations was evaluated after PA. There were no significant differences in cleavage rates. However, blastocyst (BL) formation was different. Oocytes treated with 25 mM of trehalose during IVM had a significantly higher (P < 0.05) BL formation rate (64.2%) after PA compared with the control (52.0%). The BL quality was also improved in the 25 mM trehalose-treated group. Early BL rate significantly reduced in the 25 mM trehalose-treated group as compared to control (19.6 v. 29.9%, P < 0.05). By contrast, expanded BL rate significantly increased in the 25 mM trehalose-treated group than of control (27.7 v. 11.0%, P < 0.05). Total cell numbers of BL were significantly higher (P < 0.05) in the 25 mM trehalose-treated group compared to those in the control group (52.2 v. 36.8). However, BL rate and quality of oocytes treated with 50 and 100 mM trehalose were similar with control group. In conclusion, these results indicate that 25 mM trehalose during IVM improved the developmental potential of porcine embryos. Trehalose will be useful for large-scale production of BL with good quality in porcine in vitro production.This work was supported by a grant from the Next-Generation Bio Green 21 Program (No. PJ009563032014), Rural Development Administration, Republic of Korea.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

scholarly journals N-acetyl-L-cysteine Improves the Developmental Competence of Bovine Oocytes and Embryos Cultured In Vitro by Attenuating Oxidative Damage and Apoptosis

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 860
Author(s):  
Wu-Sheng Sun ◽  
Hoon Jang ◽  
Mi-Ryung Park ◽  
Keon Bong Oh ◽  
Haesun Lee ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Early Stage ◽  
Developmental Competence ◽  
Beneficial Effects ◽  
Developmental Rates ◽  
A Cell ◽  
Bovine Oocytes ◽  
Dose Dependent

Oxidative stress has been suggested to negatively affect oocyte and embryo quality and developmental competence, resulting in failure to reach full term. In this study, we investigated the effect of N-acetyl-L-cysteine (NAC), a cell-permeating antioxidant, on developmental competence and the quality of oocytes and embryos upon supplementation (0.1–10 mM) in maturation and culture medium in vitro using slaughterhouse-derived oocytes and embryos. The results show that treating oocytes with 1.0 mM NAC for 8 h during in vitro maturation attenuated the intracellular reactive oxygen species (ROS) (p < 0.05) and upregulated intracellular glutathione levels (p < 0.01) in oocytes. Interestingly, we found that NAC affects early embryonic development, not only in a dose-dependent, but also in a stage-specific, manner. Significantly (p < 0.05) decreased cleavage rates (90.25% vs. 81.46%) were observed during the early stage (days 0–2), while significantly (p < 0.05) increased developmental rates (38.20% vs. 44.46%) were observed during the later stage (from day 3) of embryonic development. In particular, NAC supplementation decreased the proportion of apoptotic blastomeres significantly (p < 0.05), resulting in enhanced hatching capability and developmental rates during the in vitro culture of embryos. Taken together, our results suggest that NAC supplementation has beneficial effects on bovine oocytes and embryos through the prevention of apoptosis and the elimination of oxygen free radicals during maturation and culture in vitro.

scholarly journals Land Use Transitions and Farm Performance in China: A Perspective of Land Fragmentation

Land ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 792
Author(s):  
Shukun Wang ◽  
Dengwang Li ◽  
Tingting Li ◽  
Changquan Liu
Keyword(s):  
Large Scale ◽  
Household Survey ◽  
Scale Production ◽  
Land Fragmentation ◽  
Land Transfer ◽  
Large Scale Production ◽  
Speed Up ◽  
Farm Performance ◽  
Effect On Yield ◽  
The Relationship

Land fragmentation (LF) is widespread worldwide and affects farmers’ decision-making and, thus, farm performance. We used detailed household survey data at the crop level from ten provinces in China to construct four LF indicators and six farm performance indicators. We ran a set of regression models using OLS methods to analyse the relationship between LF and farm performance. The results showed that (1) LF increased the input of production material and labour costs; (2) LF reduced farmers’ purchasing of mechanical services and the efficiency of ploughing; and (3) LF may increase technical efficiency (this result, however, was not sufficiently robust and had no effect on yield). Generally speaking, LF was negatively related to farm performance. To improve farm performance, it is recommended that decision-makers speed up land transfer and land consolidation, stabilise land property rights, establish land-transfer intermediary organisations and promote large-scale production.

The potential of the smoke-derived compound 3-methyl-2H-furo[2,3-c]pyran-2-one as a priming agent for tomato seeds

2007 ◽  
Vol 17 (3) ◽  
pp. 175-181 ◽  
Author(s):  
Neeru Jain ◽  
Johannes Van Staden
Keyword(s):  
Large Scale ◽  
Seed Storage ◽  
Seed Priming ◽  
Stress Conditions ◽  
Scale Production ◽  
Seedling Vigour ◽  
Flow Cytometry Data ◽  
Tomato Seeds ◽  
Vigour Index ◽  
Continuous Presence

AbstractThe stimulatory role of 3-methyl-2H-furo[2,3-c]pyran-2-one, a smoke-derived butenolide, on germination and post-germination events is well documented. Previous studies have involved germinating seeds in the continuous presence of the compound. However, commercial growers cannot exploit the potential of the butenolide for large-scale production of crops due to limited availability and environmental constraints. The present study was undertaken to assess the potential of the butenolide as a priming agent of tomato (Solanum esculentum Mill.) seeds. Flow cytometry data revealed that butenolide-primed seeds had a higher percentage of nuclei at the 4C stage than water-primed seeds. Emergence of the radicle was much faster in the primed seeds. After 36 h of imbibition, a higher percentage of the butenolide-primed seeds (22%) exhibited radicle emergence compared to the water-primed seeds (12%). While butenolide-primed seeds initially germinated more rapidly than either water-primed or unprimed seeds in a 48-h period, water-imbibed seeds reached a similar germination level as the butenolide-primed seeds by 60 h of incubation. The butenolide-primed seeds produced significantly (P ≤ 0.05) more vigorous seedlings than water-primed seeds or seeds in the continuous presence of either water or butenolide. A gradual decrease in the seedling vigour index was recorded for both water and butenolide-primed seeds with increased seed storage at room temperature. Nevertheless, the vigour index was significantly greater in the butenolide-primed seeds than the water-primed seeds. Vigour indices were significantly (P ≤ 0.05) higher for the butenolide-primed seeds under various stress conditions (salinity, osmoticum or temperature) compared to control or water-primed seeds. Results of the present study suggest that the butenolide is a good seed-priming agent. Additionally, primed seeds retained the promotive effect for a considerable time. This was also the case for tomato seeds under various simulated field stress conditions.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

Abstract 262: Derivation of Duchenne Muscular Dystrophy Disease Specific Cardiomyocytes From Patient Urine

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Xuan Guan ◽  
David L Mack ◽  
Claudia M Moreno ◽  
Fernando Santana ◽  
Charles E Murry ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Urine ◽  
Large Scale ◽  
Lentiviral Vector ◽  
Cellular Reprogramming ◽  
Urine Samples ◽  
Pcr Analysis ◽  
Scale Production ◽  
Mechanism Study

Introduction: Human somatic cells can be reprogrammed into primitive stem cells, termed induced pluripotent stem cells (iPSCs). These iPSCs can be extensively expanded in vitro and differentiated into multiple functional cell types, enabling faithful preservation of individual’s genotype and large scale production of disease targeted cellular components. These unique cellular reagents thus hold tremendous potential in disease mechanism study, drugs screening and cell replacement therapy. Due to the genetic mutation of the protein dystrophin, many DMD patients develop fatal cardiomyopathy with no effective treatment. The underlying pathogenesis has not been fully elucidated. Hypothesis: We tested the hypothesis that iPSCs could be generated from DMD patients’ urine samples and differentiated into cardiomyocytes, recapitulating the dystrophic phenotype. Methods: iPSCs generation was achieved by introducing a lentiviral vector expressing Oct4, Sox2, c-Myc and Klf4 into cells derived from patient’s (n=1) and healthy volunteers’ (n=3) urine. Cardiomyocytes were derived by sequentially treating iPSCs with GSK3 inhibitor CHIR99021 and Wnt inhibitor IWP4. Differentiated cardiomyocytes were subjected to calcium imaging, electrophysiology recording, Polymerase Chain Reaction (PCR) analysis, and immunostaining. Results: iPSCs were efficiently generated from human urine samples and further forced to differentiate into contracting cardiomyocytes. PCR analysis and immunostaining confirmed the expression of a panel of cardiac markers. Both normal and patient iPSC derived cardiomyocytes exhibited spontaneous and field stimulated calcium transients (up to 2Hz), as well as action potentials with ventricular-like and nodal-like characteristics. Anti-dystrophin antibodies stained normal iPSC-derived cardiomyocyte membranes but did not react against DMD iPSC-derived cardiomyocytes. Conclusions: Cardiomyocytes can be efficiently generated from human urine, through the cellular reprogramming technology. DMD cardiomyocytes retained the patient’s genetic information and manifested a dystrophin-null phenotype. Functional assessments are underway to determine differences that may exist between genotypes.

Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo

Reproduction ◽  
2021 ◽  
Vol 161 (4) ◽  
pp. 353-363
Author(s):  
Mun-Hyeong Lee ◽  
Pil-Soo Jeong ◽  
Bo-Woong Sim ◽  
Hyo-Gu Kang ◽  
Min Ju Kim ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Early Phase ◽  
Inner Cell Mass ◽  
Formation Rate ◽  
Cell Mass ◽  
Female Reproductive Tract ◽  
Developmental Competence ◽  
Early Embryos ◽  
Developmental Parameters

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0–2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2–4 days) and late phases (4–6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

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