294. Insulin receptor internalization in mouse preimplantation embryos

2005 ◽  
Vol 17 (9) ◽  
pp. 124
Author(s):  
F.-C. Hung ◽  
M. Pantaleon ◽  
P. L. Kaye
Keyword(s):  
Insulin Receptor ◽  
Receptor Internalization ◽  
Preimplantation Embryos ◽  
Regulation Of Transcription ◽  
Morphological Development ◽  
Cell Stage ◽  
Cytoplasmic Staining ◽  
Morula Stage ◽  
Exon 11

The insulin receptor (IR) mediates the actions of insulin and insulin-like growth factors (IGF-I and II). Two IR isoforms result from alternate splicing of exon 11, IR-A (without exon 11) and IR-B (with exon 11). Exon 11 is 36 bp and encodes 12 amino acids (717–729) in the COOH-terminus of the IR alpha-subunit. IR-A has higher binding affinity for insulin and IGF-II than IR-B. Interestingly, IR-A is predominantly expressed in fetal tissues, peripheral nerve, brain and tumours whilst IR-B is expressed primarily in classical insulin sensitive tissues such as adult liver and muscle. Our previous studies showed that in mice, like other species, the IR is expressed throughout preimplantation development. IR-B is expressed throughout the preimplantation period, whilst IR-A is expressed following compaction. Immunofluorescent confocal microsopy using an exon11 specific antiserum revealed IR-B immunoreactivity in cell membranes of zygotes and embryos to the morula stage and concentrated in the trophectoderm of blastocysts. Previous studies have shown that insulin can have proliferative effects prior to compaction.1 Consistent with a functional IR at the 2-cell stage, insulin treatment rapidly increased cytoplasmic staining for IR-B within 5-15 min suggesting IR internalization on binding of insulin, which may be either trafficking to the nucleus for regulation of transcription or bound for degradation. Further investigations are underway to address these two options. (1)Gardner, H. G., and Kaye, P. L. (1991). Insulin increase cell numbers and morphological development in mouse pre-implantation embryos in vitro. Reprod Fertil Dev 3, 79–91.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 203-213 ◽  
Author(s):  
S. Eswari ◽  
G. Sai Kumar ◽  
G. Taru Sharma
Keyword(s):  
Culture Media ◽  
Total Cell ◽  
Inhibitory Factor ◽  
Preimplantation Embryos ◽  
Leukaemia Inhibitory Factor ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryThe objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus–oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8–16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

Development and viability of bovine preimplantation embryos after the in vitro infection with bovine herpesvirus-1 (BHV-1): immunocytochemical and ultrastructural studies

Zygote ◽  
2007 ◽  
Vol 15 (4) ◽  
pp. 307-315 ◽  
Author(s):  
A.V. Makarevich ◽  
J. Pivko ◽  
E. Kubovicova ◽  
P. Chrenek ◽  
M. Slezaková ◽  
...  
Keyword(s):  
Embryo Development ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Trypsin Treatment ◽  
Cell Organelles ◽  
Virus Like Particles ◽  
Ultrastructural Studies ◽  
Morula Stage ◽  
Cellular Organelles

SummaryThe aim of our study was to examine whether: (1) the exposure of bovine embryos to the BHV-1 virus in vitro can compromise their further development and alter the ultrastructural morphology of cellular organelles; (2) whether the zona pellucida (ZP) can be a barrier protecting embryos against infection; and (3) whether washing with trypsin after viral exposure can prevent virus penetration inside the embryo and subsequent virus-induced damages. The embryos were recovered from superovulated Holstein-Friesian donor cows on day 6 of the estrous cycle. Only compact morulas or early blastocysts were selected for experiments with virus incubation. We used the embryos either with intact ZP (either with or without trypsin washing) or embryos in which the ZP barrier was avoided by using the microinjection of a BHV-1 suspension under the ZP. ZP-intact embryos (n = 153) were exposed to BHV-1 at 106.16 TCID50/ml for 60 min, then washed in trypsin according to IETS guidelines and postincubated in synthetic oviduct fluid (SOF) medium for 48 h. Some of the embryos (n = 36) were microinjected with 20 pl of BHV-1 suspension under the ZP, the embryos were washed in SOF medium and cultured for 48 h. Embryo development was evaluated by morphological inspection, the presence of viral particles was determined both immunocytochemically, using fluorescent anti-IBR–FITC conjugate and by transmission electron microscopy (TEM) on the basis of the ultrastructure of the cellular organelles.It was found that BHV-1 exposure impairs embryo development to higher preimplantation stages independent of the presence of the ZP or the trypsin treatment step, as most of the embryos were arrested at the morula stage when compared with the control. Immunofluorescence analysis confirmed the presence of BHV-1 particles in about 75% of embryos that were passed through the trypsin treatment and in all the BHV-1-microinjected embryos. Ultrastructural analysis, using TEM, revealed the presence of virus-like particles inside the BHV-1-exposed embryos, where the trypsin washing step was omitted. Conversely, in trypsin-treated BHV-1-exposed embryos, TEM detected only the envelope-free virus-like particles adhered to pores of the ZP. The embryos that were microinjected with BHV-1 suspension showed the presence of BHV-1 particles, as well as ultrastructural alterations in cell organelles. Taken together these findings may suggest that BHV-1 infection compromises preimplantation development of bovine embryos in vitro and therefore the ZP may not be enough on its own to prevent virus-induced damage, unless it is not accompanied with trypsin washing.

The effect of nerve growth factor on nuclear progression of porcine oocytes during in vitro maturation and embryo development

2005 ◽  
Vol 53 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Ágnes Bali Papp ◽  
T. Somfai ◽  
Mabel Tartaglione ◽  
Erika Varga ◽  
J. C. Gardon
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Groups ◽  
Cell Stage ◽  
Nerve Growth ◽  
Morula Stage ◽  
And Control

The present study examined the effect of nerve growth factor (NGF) on in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development of porcine oocytes. Cumulus-oocyte complexes were cultured with or without 1.0 ng/ml NGF for 40 h. After IVF, they were cultured in vitro for 6 days. After 10 and 20 h of IVM, there was no difference in nuclear status between the NGF-treated and control oocytes. Significant differences were detected in nuclear progression of oocytes matured in the presence or absence of NGF at 30 h of culture. A higher proportion of NGF-treated oocytes were at M-II stage compared to the control. Nevertheless, at the end of the 40-h IVM period, there was no difference in the proportion of M-II stage oocytes between the NGF-treated and control groups. NGF in IVM medium did not influence the developmental competence of putative embryos. Most embryos remained at the 2- to 4-cell stage; however, a significant amount of embryos reached the morula stage both in the NGF and the control groups. These results suggest that NGF during IVM accelerates nuclear progression of porcine oocytes by enhancing the post-diakinetic events of meiosis.

scholarly journals Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage

Zygote ◽  
1994 ◽  
Vol 2 (4) ◽  
pp. 281-287 ◽  
Author(s):  
Asangla Ao ◽  
Robert P. Erickson ◽  
Robert M.L. Winston ◽  
Alan H Handysude
Keyword(s):  
Mammalian Species ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Cell Stage ◽  
Gonad Differentiation ◽  
Embryonic Genome ◽  
Human Preimplantation Embryos

SummaryGlobal activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

scholarly journals Application of a novel cell-permeable peptide-driven protein delivery in mouse blastocysts

Reproduction ◽  
2013 ◽  
Vol 146 (2) ◽  
pp. 145-153 ◽  
Author(s):  
Sojung Kwon ◽  
Areum Kwak ◽  
Hyejin Shin ◽  
Soyoung Choi ◽  
Soohyun Kim ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Hela Cells ◽  
Protein Delivery ◽  
Cancer Cell Line ◽  
Preimplantation Embryos ◽  
Cervical Cancer Cell Line ◽  
Cell Stage ◽  
Cell Permeable Peptide ◽  
Morula Stage ◽  
Punctate Pattern

Cell-permeable peptides (CPPs) mediate the delivery of macromolecules into cells. However, whether CPPs are usable in mammalian oocytes and embryos for the modulation of protein expression has not been widely investigated. We have previously designed a novel 12-mer CPP from the conserved region of the human papillomavirus L1 capsid protein. In this study, we tested whether this peptide, LDP12, effectively delivers a protein cargo to mouse oocytes and preimplantation embryos. We prepared a LDP12–EGFP fusion protein having LDP12 as an N-terminal tag. This fusion protein readily enters HeLa cells, a cervical cancer cell line. The entry of LDP12–EGFP was partially blocked by amiloride, while cytochalasin D or methyl-β-cyclodextrin slightly increased the uptake. LDP12–EGFP shows efficient transduction in mouse blastocysts, but not in oocytes, two-cell-stage, or morula-stage-preimplantation embryos. LDP12-mediated delivery of EGFP–LC3, a widely used marker of autophagic activation, is successful in HeLa cells and mouse blastocysts, as it enters cells and exhibits a signature punctate pattern. The lipidation of EGFP–LC3 also normally occurs after transduction, suggesting that the transduced protein retains the functional characteristics. Collectively, we show that LDP12-driven protein delivery is a fast and convenient method applicable to mouse blastocysts and reproductive cancer cells.

TRIM28 down-regulation on methylation imprints in bovine preimplantation embryos

Zygote ◽  
2018 ◽  
Vol 26 (6) ◽  
pp. 449-456 ◽  
Author(s):  
Xin Ma ◽  
Sheng Zhang ◽  
Meiling Zhang ◽  
Yiran Zhu ◽  
Panpan Ma ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Preimplantation Embryos ◽  
Differentially Methylated Region ◽  
Fibroblast Cells ◽  
Mrna Sequence ◽  
Cell Stage ◽  
H19 Gene

SummaryTRIM28/KAP1/TIF1β was identified as a universal transcriptional co-repressor and is critical for regulating post-fertilization methylation reprogramming in preimplantation embryos. In this study, three siRNAs (si647, si742, and si1153) were designed to target the TRIM28 mRNA sequence. After transfection of the mixture of the three siRNA (siMix) into bovine fibroblast cells, the most effective one for TRIM28 knockdown was selected. By injecting RNAi directed against TRIM28 mRNA, we found that TRIM28 knockdown in oocytes had the most effect on the H19 gene, in which differentially methylated region (DMR) methylation was almost completely absent at the 2-cell stage (1.4%), while control embryos showed 74% methylation. In addition, global H3K9me3 levels at the 2-cell stage were significantly higher in the in vitro fertilization (IVF) group than in the TRIM28 knockdown group (P<0.05). We further show that TRIM28 is highly expressed during oocyte maturation and reaches peak levels at the 2-cell stage. In contrast, at this stage, TRIM28 expression in somatic cell nuclear transfer (SCNT) embryos decreased significantly (P<0.05), suggesting that Trim28 transcripts are lost during SCNT. TRIM28 is required for the maintenance of methylation imprints in bovine preimplantation embryos, and the loss of TRIM28 during SCNT may contribute to the unfaithful maintenance of imprints in cloned embryos.

Effect of glucose in the culture medium on development of horse oocytes matured and microfertilized in vitro

1995 ◽  
Vol 7 (5) ◽  
pp. 1067 ◽  
Author(s):  
T Azuma ◽  
YH Choi ◽  
S Hochi ◽  
N Oguri
Keyword(s):  
Calcium Ionophore ◽  
Fertilization Rate ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cell Stage ◽  
Morula Stage ◽  
Fetal Bovine ◽  
Oviduct Fluid ◽  
Effect Of Glucose

The development of in-vitro matured and microfertilized horse oocytes was examined in vitro. Fertilized oocytes were produced by 20-h insemination of in-vitro matured and partially zona-removed oocytes with frozen spermatozoa that had been treated with caffeine/calcium ionophore A23187 (fertilization rate 34.2%, monospermy rate 76.9%). Embryonic development was assessed by the number of nuclei stained with Giemsa solution. In Experiment 1, a continuous 8-day culture of the microfertilized oocytes in TCM199 or modified synthetic oviduct fluid (m-SOF) supplemented with 10% fetal bovine serum or 0.1% polyvinyl alcohol (PVA) in 5% O2, 5% CO2 and 90% N2 resulted in very few embryos developing beyond the 8-cell stage. In Experiment 2, the effects of different glucose concentrations (0, 0.5, 5.5 mM) in m-SOF/PVA during Days 1-4 and Days 5-8 of culture were examined. Proportions of oocytes having more than one nucleus ranged from 17.7% to 44.7% among the combinations of glucose concentrations. Supplementation with glucose at 0.5 mM during Days 1-4 followed by 5.5 mM during Days 5-8 resulted in the best embryo development; 12/55 (21.8%) nuclei-positive oocytes developed to the 8-16-cell stage, 11 (20.0%) developed to the 17-50-cell stage, and 5 (9.1%) comprised more than 50 cells and were assumed to be at the morula stage.

68 TRANSCRIPTIONAL GENOME ACTIVATION IN CANINE EMBRYOS COLLECTED IN VIVO

2012 ◽  
Vol 24 (1) ◽  
pp. 146 ◽  
Author(s):  
S. Chastant-Maillard ◽  
C. Viaris de Lesegno ◽  
S. Thoumire ◽  
M. Chebrout ◽  
K. Reynaud
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Laser Scanning ◽  
Transcriptional Activity ◽  
Staining Intensity ◽  
Laser Scanning Confocal Microscopy ◽  
Cell Stage ◽  
Morula Stage

Early embryonic stages are supported by maternal transcripts from the oocyte cytoplasm. Progressive transcription of embryonic genome is a key step for further embryonic development, especially during in vitro culture. To date, in vitro culture from fertilization to the blastocyst stage is inefficient in the canine species. The objective of this work was to identify minor and major activation in in vivo-produced dog embryos. Ovariectomies were performed in 31 Beagle bitches from 102 to 266 h after ovulation (post-ov), precisely timed by transabdominal ultrasonography. Embryos were collected by tubal flushing with M199-Hepes and immediately transferred into transcription buffer. Transcriptional activity was evaluated through 5-bromouridine 5′-triphosphate (BrUTP) incorporation in nascent RNA, without microinjection (Aoki et al. 1997). Oocytes from anoestrus ovaries were used as positive controls. 5-Bromouridine 5′-triphosphate incorporation was revealed by immunocytochemistry (anti-bromodeoxyuridine primary antibody) and embryonic DNA was stained by ethidium homodimer-2. Staining was quantified under laser scanning confocal microscopy. Transcriptional activity was calculated as (mean nuclear intensity – cytoplasmic mean intensity) × nuclear area and expressed in arbitrary units (AU). It was compared to 1 (similar intensity in nucleus and cytoplasm; i.e. no transcriptional activity) by t-test; levels of transcriptional activity were compared between stages by variance analysis. Seventy embryos (from 7 to 21 per stage) from 31 bitches were analysed, from 2 pronuclei to morula stage. Between 28 and 125 nuclei were quantified per stage. At each stage, transcriptional activity was calculated per embryo and per nucleus. A significant transcriptional activity was detected as early as the 2 pronuclei stage (102–132 h post-ov; 1.15 ± 0.05 AU). Transcriptional activity per embryo significantly increased between the 2- and the 4-cell stage and between the 8-cell and the morula stage. In early 8-cell embryos, staining intensity of the various nuclei was markedly heterogeneous within the same embryo, all nuclei being intensively stained from the late 8-cell stage onwards. Transcriptional activity per nucleus increased also from the 2- to the 4-cell stage (respectively, 120–161 h post-ov, 1.15 ± 0.02 AU and 133–154 h post-ov, 1.35 ± 0.04 AU) until the 8-cell stage (153–225 h post-ov, 5.12 ± 0.55 AU). Transcriptional levels at these 3 stages differed significantly. It decreased between the 8-cell and the morula stage (230–266 h post-ov, 3.06 ± 0.13 AU), probably reflecting the acquisition of a selectivity in gene expression at major activation, as in other species; Nothias et al. 1995). Addition of the transcriptional inhibitor α-amanitin during BrUTP incubation decreased the transcriptional activity by 60% (P < 0.05). Embryonic gene expression (minor activation) thus begins in the canine embryo as early as the 2 pronuclei stage, with major activation taking place during the 8-cell stage.

Sign in / Sign up

Export Citation Format

Share Document