105 COMPARATIVE ULTRASTRUCTURE OF IN VITRO-PRODUCED BOVINE BLASTOCYSTS (BOS INDICUS) DERIVED FROM IN VITRO FERTILIZATION, SNC, AND PARTHENOGENESIS

2013 ◽  
Vol 25 (1) ◽  
pp. 200
Author(s):  
F. Oliveira ◽  
F. Perecin ◽  
F. Meireles ◽  
J. Sangalli ◽  
Y. Watanabe ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
First Trimester ◽  
High Rate ◽  
Normal Embryo ◽  
Vitro Fertilization ◽  
Parthenogenetic Embryos

It is known that embryos produced in vitro may have structural alterations that often compromise the normal embryo development, generating a high rate of pregnancy loss. The study of these changes is of great importance because it may elucidate the cause of embryonic loss during the first trimester of pregnancy. Thus, the objective of this study was to characterize and compare ultrastructurally bovine blastocysts in the 7th day of development produced by IVF, cloning by somatic cell nuclear transfer (SCNT), and parthenogenesis. In vitro-produced embryos were derived from in vitro-matured oocytes. The somatic cell used to make cloning was fibroblasts of adult cows, and the protocol for parthenogenetic activation of the embryos was done with ionomycin-DMAP. The blastocysts derived from the different experimental groups were fixed in 2.5% glutaraldehyde and processed for transmission electron microscopy evaluation. The results showed that blastocysts derived by SNC and parthenogenesis exhibited a significantly reduced size; the inner cell mass and the blastocoel were not well defined compared with IVF embryos, indicating a less-advanced state of development. Furthermore, organelles of blastocysts derived from SCNT and parthenogenesis were fewer in number and had changes in form, when compared with IVF blastocysts. In parthenogenetic embryos there was the presence of phagosomes, suggesting a high degradation activity of cellular. Mitochondria showed the most significant changes. Although they occur in large quantities in all blastocysts, the morphology of them was impaired in SNC and parthenogenetic embryos (vacuolization, abnormal shape). Such modifications could suggest changes in mitochondria functionality, which may decrease cellular metabolic activity. Thus, we find that the D7 blastocysts derived from SCNT and parthenogenesis showed several ultrastructural differences compared with IVF embryos, with particular reference to a reduced number and morphology of embryo organelles.

scholarly journals Effect of follicular diameter, time of first cleavage and H3K4 methylation on embryo production rates of Bos indicus cattle

2016 ◽  
Vol 37 (5) ◽  
pp. 3189
Author(s):  
Paula Alvares Lunardelli ◽  
Luciana Simões Rafagnin Marinho ◽  
Camila Oliveira Rosa ◽  
Amauri Alcindo Alfieri ◽  
Marcelo Marcondes Seneda
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Embryo Viability ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Trophectoderm Cells ◽  
Preimplantation Stage ◽  
First Time

This study aimed investigate the relationship between epigenetics, follicular diameter and cleavage speed, by evaluating the developmental potential and occurence of H3K4 monomethylation of early-, intermediate- and late-cleaving Bos indicus embryos from in vitro fertilized oocytes originating from follicles up to 2 mm in diameter or between 4 and 8 mm in diameter. Oocytes (n = 699) from small follicles (? 2 mm) and 639 oocytes from large follicles (4-8 mm) were punched from 1,982 Bos indicus’ slaughterhouse ovaries. After maturation and in vitro fertilization (IVF), the cultured embryos were separated into early (? 28 h post-IVF), intermediate (> 28 h and ? 34 h post-IVF) and late (> 34 h and ? 54 h post-IVF) cleavage groups. Blastocysts were subjected to an immunofluorescence assessment for H3K4me investigation. The blastocyst rate for large follicles (36.3%) was higher than that for small follicles (22.9%, P < 0.05). In addition, blastocyst rates for early and intermediate cleavage groups (45.3% and 33.8%, respectively) were higher than that for late cleavage group (13.5%, P < 0.05). The blastocysts from all groups displayed H3K4me staining by immunofluorescence, particularly intense in what seemed to be trophectoderm cells and weak or absent in cells seemingly from the inner cell mass. For the first time for indicus embryos, data from this study demonstrate that higher blastocyst embryo rates are obtained from embryos that cleave within 34 h after fertilization and from those produced from follicles of 4-8 mm in diameter, indicating a greater ability of these embryos to develop to the stage of embryonic preimplantation. This is the first article demonstrating the occurrence of H3K4me in cattle embryos; its presence in all the evaluated blastocysts suggests that this histone modification plays a key role in maintaining embryo viability at preimplantation stage.

CSF-1 and mouse preimplantation development in vitro

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1333-1339 ◽  
Author(s):  
P. Bhatnagar ◽  
V.E. Papaioannou ◽  
J.D. Biggers
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Preimplantation Development ◽  
High Rate ◽  
Colony Stimulating Factor ◽  
Trophoblast Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The effects of macrophage colony stimulating factor on the development of the zygote to the blastocyst stage of an outbred strain of mouse have been studied in KSOM, an improved medium that supports a high rate of in vitro development. Macrophage colony stimulating factor accelerates the formation of the blastocyst cavity by day 4 (96 hours post-hCG). It also increases overall embryonic cell number through a differential increase in the number of trophoblast cells, with no significant effect on the number of inner cell mass cells. By day 5 of culture (120 hours post-hCG), colony stimulating factor-treated embryos have about 20 more trophoblast cells than control embryos, an increase of about 30 percent of the total number of cells in a control blastocyst. The maximum response of embryos was obtained at a concentration around 540 U ml-1 colony stimulating factor (identical to 918 Stanley units ml-1), and the cytokine can produce the same effects even if it is present in the medium for only part of the culture period. This in vitro stimulation of preimplantation development with macrophage colony stimulating factor is compatible with continued normal fetal development in vivo.

scholarly journals In vitro fertilization and culture alters chromatin accessibility in the mouse inner cell mass

2018 ◽  
Vol 110 (4) ◽  
pp. e378
Author(s):  
E. Ruggeri ◽  
E. Grow ◽  
X. Liu ◽  
A. Donjacour ◽  
P. Rinaudo
Keyword(s):  
In Vitro Fertilization ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Chromatin Accessibility ◽  
Vitro Fertilization

53 EFFECTS OF INSULIN-TRANSFERRIN-SELENIUM IN DEFINED AND PORCINE FOLLICULAR FLUID-SUPPLEMENTED MEDIUM ON IN VITRO PRODUCTION OF PORCINE SCNT EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
Y. U. Kim ◽  
D. P. Bhandari ◽  
M. S. Hossein ◽  
S. M. Park ◽  
E. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Defined Medium ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Differential Staining ◽  
In Vitro Production ◽  
Fetal Fibroblasts

Insulin promotes the uptake of glucose and amino acids, and is beneficial for maturation of oocytes in vitro. Transferrin is an iron-transport protein and selenium is an essential trace element. Insulin-transferrin-selenium (ITS) together has been used in some in vitro maturation systems. The present study was designed to evaluate the effects of ITS in defined and porcine folicular fluid (pFF)-supplemented IVM medium on the glutathione (GSH) concentration, and on developmental competence after somatic cell nuclear transfer. ITS liquid media supplement (I-3146) was purchased from Sigma-Aldrich (St Louis, MO, USA). Basic IVM medium was TCM-199 supplemented with 10 ng mL-1 epidermal growth factor, 4 IU mL-1 pregnant mare serum gonadotropin (PMSG) and hCG and either 1% PVA (defined medium) or 10% pFF. Ten �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium was used for the entire 44-h culture period. The GSH content of a gruop of 10 to 20 oocytes was determined by the dithionitrobezoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Fetal fibroblasts were used as somatic cell donors and reconstructed embryos were cultured in mNCSU-23 medium for 168 h. Cleavage and blastocyst formation was observed at 48 h and 168 h, respectively. The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM) and the trophectoderm (TE) cells. Each experiment was replicated for 5 times. The data were analyzed by one-way ANOVA, and Tukey was used as a posthoc test. The level of GSH production significantly varied in different culture conditions. The highest GSH concentration was observed in the pFF + ITS group (8.2 picomol/oocyte). A total of 116, 125, 126, and 120 reconstructed oocytes were cultured, and 10.1, 15.3, 17.2, and 21.8% blastocysts were observed for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P &lt; 0.05). The numbers of inner cell mass, trophrectoderm cells, and total cells were significantly higher in the pFF + ITS group compared with the other groups. The average number of total cells in blastocysts was 31.9 � 1.8, 43.1 � 3.5, 46.7 � 4.9, and 52.3 � 6.7 for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P &lt; 0.05). ITS supplement improved the developmental competence in both the defined and the pFF supplemented groups. We recommend supplementing porcine IVM medium with 10 �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium.

scholarly journals Effect of serum-containing and serum-free culture medium-mediated activation of matrix metalloproteinases on embryonic developmental competence

2019 ◽  
Vol 64 (No. 12) ◽  
pp. 473-482
Author(s):  
Sang Hwan Kim ◽  
Jong Taek Yoon
Keyword(s):  
Embryonic Development ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Competence ◽  
Serum Free Medium ◽  
Vitro Fertilization ◽  
Serum Free ◽  
Foetal Bovine Serum

In this study, we examined whether serum-free and serum-containing media affect matrix metalloproteinase (MMP) activity with respect to embryonic development, and whether MMP expression is correlated with the development of in vitro fertilized eggs. When oocytes were cultured in serum-free medium (containing polyvinylpyrrolidone) and serum (foetal bovine serum)-containing medium, the generation of meiosis 2 (MII) oocytes was 76% and 87.5%, respectively (P &lt; 0.05). After in vitro fertilization using mature oocytes, we observed 39.72% and 64.05% of cleaved oocytes in serum-free and serum-containing groups, respectively (P &lt; 0.05). Our analysis revealed differential expression and activity of MMPs. The serum-containing group showed high MMP-9 activity during oocyte maturation and development of in vitro produced embryos, with particularly high activity in the inner cell mass zone of the embryos. Therefore, this study suggests that the presence or the absence of serum will affect the activity of MMPs, which can be used to measure the rate of embryonic development.

scholarly journals OCT4/POU5F1 is required for NANOG expression in bovine blastocysts

2018 ◽  
Vol 115 (11) ◽  
pp. 2770-2775 ◽  
Author(s):  
Kilian Simmet ◽  
Valeri Zakhartchenko ◽  
Julia Philippou-Massier ◽  
Helmut Blum ◽  
Nikolai Klymiuk ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Preimplantation Development ◽  
Null Mouse ◽  
General Strategy ◽  
Vitro Fertilization ◽  
Domestic Species ◽  
Oct4 Protein ◽  
Salt And Pepper

Mammalian preimplantation development involves two lineage specifications: first, the CDX2-expressing trophectoderm (TE) and a pluripotent inner cell mass (ICM) are separated during blastocyst formation. Second, the pluripotent epiblast (EPI; expressing NANOG) and the differentiated primitive endoderm (PrE; expressing GATA6) diverge within the ICM. Studies in mice revealed that OCT4/POU5F1 is at the center of a pluripotency regulatory network. To study the role of OCT4 in bovine preimplantation development, we generated OCT4 knockout (KO) fibroblasts by CRISPR-Cas9 and produced embryos by somatic cell nuclear transfer (SCNT). SCNT embryos from nontransfected fibroblasts and embryos produced by in vitro fertilization served as controls. In OCT4 KO morulae (day 5), ∼70% of the nuclei were OCT4 positive, indicating that maternal OCT4 mRNA partially maintains OCT4 protein expression during early development. In contrast, OCT4 KO blastocysts (day 7) lacked OCT4 protein entirely. CDX2 was detected only in TE cells; OCT4 is thus not required to suppress CDX2 in the ICM. Control blastocysts showed a typical salt-and-pepper distribution of NANOG- and GATA6-positive cells in the ICM. In contrast, NANOG was absent or very faint in the ICM of OCT4 KO blastocysts, and no cells expressing exclusively NANOG were observed. This mimics findings in OCT4-deficient human blastocysts but is in sharp contrast to Oct4-null mouse blastocysts, where NANOG persists and PrE development fails. Our study supports bovine embryogenesis as a model for early human development and exemplifies a general strategy for studying the roles of specific genes in embryos of domestic species.

scholarly journals 71 DEVELOPMENTAL DELAY OF PRE-IMPLANTATION OVINE IN VITRO CULTURED AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 185
Author(s):  
P. Tveden-Nyborg ◽  
T. Peura ◽  
K. Hartwich ◽  
P. Maddox-Hyttel
Keyword(s):  
Somatic Cell ◽  
Developmental Delay ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Germ Layers ◽  
Visceral Mesoderm

Despite advances in the production of somatic cell nuclear transfer (SCNT) embryos, significant embryo losses are persistent, particularly around implantation. Malformations of the placenta and in a variety of organs are seen, and have been linked to deviant epigenetic reprogramming. The aim of the present study was to examine the formation of germ layers, which are prerequisites for formation of the embryo proper and placenta, in invivo-derived (in vivo), partly in vitro-cultured (IVC), and SCNT ovine embryos. Embryos were derived as follows: In vivo embryos (n = 27) were flushed from the uterus on Days 7, 9, 11, and 13. For IVC embryos (n = 22) in vivo zygotes were flushed, followed by culture in the presence of 20% human serum, transfer to the uterus on Day 6, and flushing as in vivo embryos. SCNT embryos (n = 41) were produced by fusion of serum starved granulosa cells with enucleated oocytes, followed by activation, culture in SOF, transfer to the uterus on Day 6, and flushing as described for in vivo embryos. Recovered embryos were processed for light microscopy (LM) and transmission electron microscopy (TEM), and paraffin sections were immunohistochemically labelled for the germ layers: alpha-1-fetoprotein for potential endoderm, cytokeratin-8 for potential ectoderm, and vimentin for potential mesoderm. A consistent delay of the IVC and particularly the SCNT embryos was noted throughout all time points: On Days 7 and 9, differentiation of the inner cell mass into hypoblast and epiblast was evident in 7 out of 12 in vivo embryos, whereas this phenomenon was less prominent or absent in 9 out of 13 IVC and 13 out of 15 SCNT embryos. Furthermore, 6 of the IVC and 12 of the SCNT embryos lacked an identifiable embryonic disc. On Day 11, half of the in vivo embryos had initiated gastrulation, evidenced by localization of endoderm and mesoderm precursor cells between the hypoblast and the epiblast. This feature was noted in only a single IVC and in none of the SCNT embryos. On Day 13, all in vivo embryos had completed gastrulation including the formation of somatic and visceral mesoderm. This feature was noted in only 1 out of 3 IVC and in none of the SCNT embryos. Likewise, amniotic folds were seen in one third of the in vivo embryos at this stage, but not observed in any IVC or SCNT embryos. The immunohistochemical markers displayed the same cell lineage localization in all three groups of embryos, but a developmental delay in the IVC and in particular the SCNT embryos was evident. In conclusion, ovine IVC and SCNT embryos develop at a slower rate than in vivo embryos at least up until Day 13 of gestation.

275 DERIVATION OF PLURIPOTENT CELL LINES FROM PIG EMBRYOS: IN VITRO-FERTILIZED V. PARTHENOGENTIC ACTIVATION

2009 ◽  
Vol 21 (1) ◽  
pp. 235
Author(s):  
F. Gandolfi ◽  
G. Pennarossa ◽  
L. Attanasio ◽  
S. Antonini ◽  
B. Gasparrini ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Telomerase Activity ◽  
Animal Biotechnology ◽  
Pluripotency Markers ◽  
Biology Research ◽  
Parthenogenetic Embryos

The establishment of porcine pluripotent ES cell lines would be an exciting and novel tool for animal biotechnology, such as cloning and transgenesis. Furthermore, it would represent a useful model for biomedical research, cell therapy, xenotransplantation as well as developmental biology research. However, in spite of several studies, no conclusive results have been obtained and a number of technical questions are still to be answered in order to derive genuine ES cells in the pig. Here we report the results obtained in our laboratory aimed at comparing IVF v. parthenogenetic embryos as a source for the establishment of putative ES cells. Oocytes were divided in two groups and subjected to IVF or parthenogenetically activated with ionomicyn and 6-DMAP. They were cultured in NCSU for 7 days and then subjected to immuno-surgery. Inner cell mass were plated onto inactivated STO feeder cells and outgrowth formation was monitored. Cells were passaged to a new STO monolayer every 7 days. Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 22 passages. Telomerase activity was measured every 5 passages. The results indicate that parthenogenetic embryos, although less resilient than IVF embryos to immuno-surgery, have a significantly greater ability to generate outgrowths and stable cell lines. Moreover, 77% of the 39 parthenogenetic lines derived v. only 33% of the IVF ones expressed pluripotency markers and displayed high telomerase activity. Altogether our findings are consistent with data obtained in the human where the efficiency to derive hES cell lines from parthenogenetic blastocysts appears greater as compared with regular blastocysts from IVF embryos (Cheng L 2008 Cell Research 18, 215–217). Table 1. Supported by: Prin 2005, 2006.

143 TRANSCRIPTIONAL PROFILING BY HIGH-THROUGHPUT SEQUENCING OF PORCINE PRE- AND PERI-IMPLANTATION EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 184 ◽  
Author(s):  
S. C. Isom ◽  
J. R. Stevens ◽  
R. Li ◽  
L. D. Spate ◽  
W. G. Spollen ◽  
...  
Keyword(s):  
Somatic Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Assisted Reproductive Technologies ◽  
Inner Cell Mass ◽  
Expression Profiles ◽  
Cell Mass ◽  
Reproductive Technologies ◽  
Regulation Of Transcription

Significant embryo mortality occurs at or around the time of implantation or attachment in virtually all mammalian species studied to date, even in naturally conceived embryos. Embryos resulting from assisted reproductive technologies (ART) are even more susceptible to peri-implantation failure. Herein we describe our effort to characterise the transcriptomes of embryonic disc (ED) and trophoblast (TE) cells from porcine embryos derived from AI, IVF, parthenogenetic oocyte activation (PA) and somatic cell nuclear transfer (NT) on Days 10, 12 and 14 of gestation. The IVF, PA and somatic cell NT embryos were generated using in vitro–matured oocytes, cultured overnight in vitro and then transferred at the 1- to 2-cell stage into appropriately synchronized recipient gilts. On the appropriate collection day, embryos were flushed from the uterus and ED was separated from TE by mechanical dissection. Double-stranded cDNA from the collected samples was sequenced using the GAII platform from Illumina (San Diego, CA, USA). The resulting sequencing reads were aligned to a custom swine transcriptome database (see Isom et al. 2010). A generalized linear model was fit for each of 41 693 genomic regions, for ED and TE samples separately, accounting for embryo type, gestation day and their interaction and using total lane read count as a normalizing offset. Genes with significant embryo type differences (controlling the false discovery rate at 0.10) were subsequently tested for differences between IVF and each of AI, PA and NT. Those genes with significant post hoc differences (either up- or down-regulated compared with IVF) were characterised in terms of gene ontologies and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using a gene set enrichment test. Bone morphogenetic protein signalling was down-regulated (KEGG; P = 0.0099; adjusted to control for FDR at 0.05) in the ED of IVF embryos when compared with AI embryos. In TE cells from IVF embryos, ubiquitin-mediated proteolysis and ErbB signalling (adj P = 0.031 for both pathways) were aberrantly regulated when compared with AI embryos. Of particular interest is the observation that expression of genes involved in chromatin modification (GO:BiologicalProcess; q-value = 0.00005) and epigenetic regulation of transcription (q = 0.00007) was very significantly disrupted in inner cell mass cells from NT embryos compared with IVF embryos. Surprisingly, no such disruption of the epigenetic machinery was observed in the TE cells from NT embryos. In summary, we have used high-throughput sequencing technologies to compare gene expression profiles of various ART embryo types during peri-implantation development. We expect that these data will provide important insight into the root causes of (and possible opportunities for mitigation of) suboptimal development of embryos derived from ART. Funding was received from NIH R01 RR013438 and Food for the 21st Century (RSP) and the Utah Agricultural Experiment Station (UTA00151 and UTA00560 for S. C. Isom and J. R. Stevens, respectively).

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