Intracellular localisation of platelet-activating factor during mammalian embryo development in vitro: a comparison of cattle, mouse and human

2019 ◽  
Vol 31 (4) ◽  
pp. 658
Author(s):  
L. T. M. Vandenberghe ◽  
B. Heindryckx ◽  
K. Smits ◽  
M. Popovic ◽  
K. Szymanska ◽  
...  
Keyword(s):  
Platelet Activating Factor ◽  
Preimplantation Embryos ◽  
Mammalian Embryo ◽  
Developmental Potential ◽  
Nuclear Envelope Breakdown ◽  
Cytosolic Phospholipase ◽  
Carrier Molecule ◽  
Development In Vitro ◽  
Intracellular Localisation

Platelet-activating factor (PAF) is a well-known marker for embryo quality and viability. For the first time, we describe an intracellular localisation of PAF in oocytes and embryos of cattle, mice and humans. We showed that PAF is represented in the nucleus, a signal that was lost upon nuclear envelope breakdown. This process was confirmed by treating the embryos with nocodazole, a spindle-disrupting agent that, as such, arrests the embryo in mitosis, and by microinjecting a PAF-specific antibody in bovine MII oocytes. The latter resulted in the absence of nuclear PAF in the pronuclei of the zygote and reduced further developmental potential. Previous research indicates that PAF is released and taken up from the culture medium by preimplantation embryos invitro, in which bovine serum albumin (BSA) serves as a crucial carrier molecule. In the present study we demonstrated that nuclear PAF does not originate from an extracellular source because embryos cultured in polyvinylpyrrolidone or BSA showed similar levels of PAF in their nuclei. Instead, our experiments indicate that cytosolic phospholipase A2 (cPLA2) is likely to be involved in the intracellular production of PAF, because treatment with arachidonyl trifluoromethyl ketone (AACOCF3), a specific cPLA2 inhibitor, clearly lowered PAF levels in the nuclei of bovine embryos.

Metabolic and developmental responses of preimplantation embryos to platelet activating factor (PAF)

1992 ◽  
Vol 4 (4) ◽  
pp. 387 ◽  
Author(s):  
JP Ryan ◽  
C O'Neill ◽  
AJ Ammit ◽  
CG Roberts
Keyword(s):  
Culture Media ◽  
Platelet Activating Factor ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Mouse Bioassay ◽  
Limiting Factor ◽  
Cell Stage ◽  
Culture In Vitro ◽  
Developmental Potential

Platelet activating factor (PAF) is an ether phospholipid produced by preimplantation embryos of a number of species. Production of PAF by embryos has been measured by detecting thrombocytopenia in a splenectomized mouse bioassay, platelet aggregation bioassays in vitro and a specific radioimmunoassay. Production is highly variable and is adversely affected by culture in vitro. It has, however, been correlated with morphology, development rates in vitro and the pregnancy potential of embryos following transfer. Investigations using PAF-antagonists have established an essential role for PAF in early pregnancy. Together with studies that have shown PAF to have direct effects on embryonic metabolism during culture in vitro, these observations suggest that PAF acts as an embryonic autocoid. Hence, a major site of action for embryo-derived PAF in vivo is the embryo itself. Supplementation of embryo culture media with PAF had no effect on the rate of development in vitro of 2-cell mouse embryos through to the blastocyst stage. However, PAF increased cell numbers of blastocysts cultured from the 2-cell stage and the mitotic index of embryos at both the 8-cell and blastocyst stages. Supplementation of culture media with PAF has also been shown to increase the implantation potential of both mouse and human embryos cultured in vitro. In the mouse, the effect of PAF in enhancing implantation rates was most evident when the developmental potential of untreated embryos was suboptimal. These observations suggest that the production of embryo-derived PAF is one limiting factor in maintaining the viability of embryos cultured in vitro.

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Author(s):  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

scholarly journals A uterus-inspired 3D niche drives embryo development beyond implantation

2020 ◽  
Author(s):  
Zhen Gu ◽  
Jia Guo ◽  
Jinglei Zhai ◽  
Guihai Feng ◽  
Xianning Wang ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Developmental Process ◽  
High Rate ◽  
Mammalian Embryo ◽  
Uterine Endometrium ◽  
Experimental Systems ◽  
In Vitro Systems ◽  
Development In Vitro

Abstract The mammalian embryo must undergo dramatic morphogenetic changes to invade the uterine endometrium and achieve implantation. Thus, recapitulation of implantation using in vitro systems is crucial for revealing the mechanisms controlling early development and the main problems compromising human fertility. Experimental systems based on two-dimensional (2D) platforms cannot fully recapitulate the in vivo 3D microenvironments of the embryo. Therefore, here we use collagen grafted onto polydimethylsiloxane (PDMS) based on the uterine mechanics and microstructure to establish a uterus-inspired 3D niche (U3N). Our U3N enables mouse embryos to form egg cylinders at high rate and reach the developmental stages of heartbeat. Moreover, a unique interface forms between the embryo and collagen, showing the invasion of trophoblasts into collagen fires, which simulate the developmental process of implantation. Our findings highlight embryo-substrate interaction as a key characteristic of post-implantation development in vitro and as an important design parameter of 3D conditions for embryo culture.

45 DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS DERIVED FROM ADULT MARROW STROMAL CELLS AND FETAL MUSCLE CELLS

2009 ◽  
Vol 21 (1) ◽  
pp. 122
Author(s):  
M. Murakami ◽  
X. J. Bai ◽  
W. S. Shi ◽  
W. M. Wang ◽  
W. Liu ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Donor Cell ◽  
Muscle Cells ◽  
Marrow Stromal Cells ◽  
Developmental Potential ◽  
Development In Vitro ◽  
Adult Mscs ◽  
Fetal Muscle

The use of less differentiated cells, such as marrow stromal cells (MSCs), as the nuclear donor may increase the efficiencies of somatic cell cloning in cattle. Healthy offspring was produced from bovine MSCs (Kato et al. 2004 Biol. Reprod. 70, 415–418); however, there is little information that directly compared the post-implantation survival among the clones originated from MSCs and other somatic cells. The objective of this study was to evaluate the developmental potential in vitro and in vivo of bovine NT embryos derived from adult MSCs and fetal muscle cells (FMCs). Primary cell populations of MSCs and FMCs were obtained from the femurs of 8- and 12-months-old Holstein cows (MSC1 and MSC2 groups, respectively) and a Holstein fetus at 8 months of gestation (FMC group), respectively. They were used as donor cells for the NT procedure (Murakami et al. 2005 Cloning Stem Cells 7, 77–81) at passages 1 to 3. Briefly, oocytes collected from cow ovaries were enucleated at 20 h post-in vitro maturation (IVM), and the donor cell was placed into the perivitelline space. The couplets were fused electrically, activated (10 μg mL–1 cycloheximide; 4 h), and cultured in CR1aa medium. Development in vitro of these embryos is summarized in Table 1. Data were analyzed by ANOVA. The fusion rates were higher in the MSC groups than in the FMC group. The rate of cleaved embryos was significantly lower (P < 0.05) in the MSC1 group than in the other groups. However, there were no significant differences among the groups in the rates of development into morulae/blastocysts on Day 6. A total of 8 and 3 fresh good quality Day 6 embryos in the MSC1 and FMC groups, respectively, were nonsurgically transferred to 6 naturally cycling Holstein females 6 days after estrus (3 recipients/group, 1–3 embryos/female). On Day 30 of gestation, none of the recipients were pregnant in the FMC group, while 2 recipients in the MSC1 group were diagnosed as pregnant via ultrasonography; they remained pregnant on Day 80 of gestation. In addition, a total of 4 Day 7 embryos cryopreserved in 1.8 m ethylene glycol plus 0.05 m trehalose were directly transferred to 4 synchronized recipients after thawing (1 embryo/female) in the MSC1 group. Of those, 2 females were pregnant on day 30 of gestation. These results indicate that the developmental potential in vitro of bovine NT embryos derived from adult MSCs was comparable to that of the embryos derived from fetal muscle cells, and that pregnancies were produced after transfer of the fresh and frozen–thawed NT embryos derived from the MSC, but the sample size was small. Further studies with more replicates are needed to evaluate viability in vivo of these cloned embryos for comparative purposes. Table 1.Development in vitro of bovine NT embryos derived from different cell types

210 CLEAVAGE ASSESSMENT AT DAY 2 TO PREDICT BLASTOCYST DEVELOPMENTAL POTENTIAL IN PORCINE IN VITRO-FERTILIZED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 203
Author(s):  
Y. J. Kim ◽  
Y. P. Jeon ◽  
S. H. Hyun
Keyword(s):  
Cumulus Cells ◽  
Preimplantation Development ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Embryo Morphology ◽  
Blastocyst Formation ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
The Relationship

Porcine embryos could be a valuable tool to study preimplantation development, implantation, and pregnancy, but to do this it is necessary to establish an efficient in vitro embryo production system. Because the cause of high mortality in embryos during preimplantation development is not clear, a noninvasive method of determining the developmental potential of cleavage-stage embryos is needed. The objective was to evaluate the developmental potential of Day 2 embryos in a porcine in vitro fertilization (IVF) system. Specifically, this study was conducted to examine the relationship between embryo morphology 48 h after IVF on rates of blastocyst formation 5 days later. To prepare in vitro maturation (IVM) of porcine oocytes, cumulus–oocyte complexes were obtained from slaughterhouse-derived ovaries and matured in M-199 medium supplemented with 10% pig follicular fluid and 0.57 mm cysteine for 44 h and then freed from cumulus cells. After IVM, cumulus-free oocytes were coincubated with frozen–thawed sperm (2 × 106 cells mL–1) and 2 mm caffeine for 6 h. Inseminated embryos were cultured in NCSU-23 medium that was supplemented with 0.5 mm pyruvate and 0.5 mm lactate. Data were analyzed by ANOVA and Duncan’s test (P < 0.05). Morphology data on a total of 919 embryos were analyzed retrospectively. Forty-eight hours after insemination, embryos were classified into the following 5 groups based on the cleavage state: 1 cell, 2 cells, 4 cells, 5 to 8 cells, and fragmentation. These groups were cultured another 120 h and then evaluated for blastocyst formation. Blastocyst formation rates were significantly higher in the 4-cell (38.07%) and 5- to 8-cell (40.65%) cleaving groups than in the other groups (P < 0.05). In contrast, the 2-cell and fragmentation groups produced 7.5 and 2.9% blastocysts, respectively. Data suggest that embryos reaching 4 cells and 5 to 8 cells by 48 h after insemination have high developmental competence, and this parameter may be useful to predict the development of preimplantation embryos and their ability to establish pregnancy. This work was supported by a grant (No. 20070301034040) from the BioGreen 21 program, Rural Development Administration, Republic of Korea.

De novo transcription of thyroid hormone receptors is essential for early bovine embryo development in vitro

2018 ◽  
Vol 30 (5) ◽  
pp. 779 ◽  
Author(s):  
N.-Y. Rho ◽  
F. A. Ashkar ◽  
T. Revay ◽  
P. Madan ◽  
G.-J. Rho ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Embryo Development ◽  
De Novo ◽  
Preimplantation Embryos ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Protein Levels ◽  
Development In Vitro

Thyroid hormone receptor (THR) α and THRβ mediate the genomic action of thyroid hormones (THs) that affect bovine embryo development. However, little is known about THRs in the preimplantation embryo. The aim of the present study was to investigate the importance of THRs in in vitro preimplantation bovine embryos. THR transcripts and protein levels were detected in developing preimplantation embryos up to the blastocyst stage. Embryonic transcription of THRs was inhibited by α-amanitin supplementation, and both maternal and embryonic transcription were knocked down by short interference (si) RNA microinjection. In the control group, mRNA and protein levels of THRs increased after fertilisation. In contrast, in both the transcription inhibition and knockdown groups there were significant (P < 0.05) decreases in mRNA expression of THRs from the 2-cell stage onwards. However, protein levels of THRs were not altered at 2-cell stage, although they did exhibit a significant (P < 0.05) decrease from the 4-cell stage. Moreover, inhibition of de novo transcripts of THRs using siRNA led to a significant (P < 0.01) decrease in the developmental rate and cell number, as well as inducing a change in embryo morphology. In conclusion, THRs are transcribed soon after fertilisation, before major activation of the embryonic genome, and they are essential for bovine embryo development in vitro.

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