scholarly journals K+ efflux through two-pore domain K+ channels is required for mouse embryonic development

Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 625-636 ◽  
Author(s):  
Chang-Gi Hur ◽  
Eun-Jin Kim ◽  
Seong-Keun Cho ◽  
Young-Woo Cho ◽  
Sook-Young Yoon ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mammalian Cells ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Blastocyst Stage ◽  
Ruthenium Red ◽  
Channel Blockers ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Wide Range ◽  
Morula Stage

Numerous studies have suggested that K+ channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K+ channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K+ channel blockers to identify the functional role of K+ channels in mouse embryonic development. Voltage-dependent K+ channel blockers, such as tetraethylammonium and BaCl2, had no effect on embryonic development to the blastocyst stage, whereas K2P channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K2P channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ∼38% compared with scrambled siRNA injection (P<0.05). The blockade of K2P channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K2P channels could improve mouse embryonic development through the modulation of gating by activators.

190 EXPRESSION OF GENES ASSOCIATED WITH WINGLESS-RELATED MOUSE MAMMARY TUMOUR VIRUS SIGNALING IN THE PRE-IMPLANTATION BOVINE EMBRYO

2015 ◽  
Vol 27 (1) ◽  
pp. 186
Author(s):  
P. Tribulo ◽  
J. I. Moss ◽  
P. J. Hansen
Keyword(s):  
Embryonic Development ◽  
Blastocyst Stage ◽  
Wnt Signalling ◽  
Mammary Tumour ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Mouse Mammary Tumour Virus ◽  
Ct Value ◽  
Morula Stage ◽  
Canonical Wnt

Wingless-related mouse mammary tumour virus (WNT) signalling participates in early embryonic development to maintain pluripotency, controls cell–cell communication, and modulates cell polarization and migration. To gain an understanding of the regulation of WNT signalling during embryonic development, expression patterns of a variety of molecules involved in WNT signal transduction were evaluated. Specific genes were DKK1, an endogenous inhibitor of canonical WNT signalling, the WNT co-receptors LRP5 and LRP6, WNT-responsive transcription factors, LEF1 and TCF7, and two repressors of WNT-regulated genes, the bovine orthologue of GROUCHO (LOC505120) and AES. Embryos were produced in vitro from oocytes obtained from ovaries collected at a local abattoir. Following oocyte maturation, fertilization was performed with sperm pooled from three randomly selected bulls; a different pool of bulls was used for each replicate. Groups of 30 matured oocytes or embryos at the 2-cell [28–32 h post-insemination (hpi)], 3–4 cell (44–48 hpi), 5–8 cell (50–55 hpi), 9–16 cell (72–75 hpi), morula (120–123 hpi), and blastocyst (168–171 hpi) stages were collected. The zona pellucida was removed with proteinase, RNA was purified, cDNA synthesised using random hexamer primers and real-time qPCR performed. Data analysed were ΔCT values, which were calculated by subtracting the CT value of the geometric mean of the three housekeeping genes (GAPDH, YWHAZ, and SDHA) from the CT value of the sample. The relative transcript abundance was calculated as the 2ΔCT. Data were analysed by least-squares ANOVA using the Proc GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). A total of 5 replicates were analysed for each developmental stage. Results show significant effects of stage of development for each gene that ranged from P = 0.004 for LRP5 to P ≤ 0.0001 for AES, DKK1, LEF, LOC505120, LRP6, and TCF7. In all cases, expression declined as development advanced. Except for AES, lowest expression occurred at the blastocyst stage. Lowest expression for AES was at the morula stage; expression remained low at the blastocyst stage. For two genes, DKK1 and LEF1, there was no detectable expression at the blastocyst stage. The timing of decline in expression varied between genes, first occurring at the 9–16-cell stage (AES, LEF1, and LOC505120) or morula stage (DKK1, LRP5, LRP6, or TCF7). For DKK1, LEF1, and LRP6, there was also a slight increase in expression from the oocyte to two-cell stage. Results suggest that canonical WNT signalling is reduced at the morula and blastocyst stages relative to earlier stages in development. Research was supported by USDA-NIFA 2011-67015-30688.

scholarly journals 144 ROLE OF GnRH ON MOUSE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 222
Author(s):  
M. Montagner ◽  
A. Cropp ◽  
J. Swanson ◽  
R. Cederberg ◽  
P. Goncalves ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Gnrh Agonist ◽  
Critical Role ◽  
Blastocyst Stage ◽  
Dependent Manner ◽  
Blastocyst Formation ◽  
Morula Stage ◽  
Development In Vitro ◽  
Human Uterine Endometrium

The interaction between GnRH and its receptor on gonadotropes within the anterior pituitary gland represents a key point for regulation of the reproduction. In addition, GnRH can act in multiple extrapituitary tissues via autocrine/paracrine mechanisms. Protein for GnRH and mRNA for both GnRH and its receptor have been detected in human uterine endometrium and oviduct as well as in embryos at the morula/blastocyst stage in the mouse and human. Therefore, we hypothesized that GnRH may have a critical role in the development of pre-implantation embryos. To address this question, we examined the effect of a GnRH agonist and antagonist on the development of mouse embryos in vitro. For these studies, 1-cell embryos were randomly allocated to culture in KSOM containing the appropriate treatment for 144 h at 37°C in a 5% CO2 in air environment. The medium was changed every 12 h and embryos were scored daily for development. The data were compared using a χ2 test. First, we wanted to determine if a GnRH agonist, histrelin, could enhance embryonic development. Embryos were cultured with (n = 35) or without (n = 36) 10 μM histrelin. The addition of histrelin did not increase morula or blastocyst formation v. control. Second, we cultured embryos in the presence of different concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μM) of the GnRH antagonist, SB-75 (cetrorelix; n = 22/treatment) in order to determine its effect on embryonic development. The 10 μM SB-75 treatment blocked embryo development beyond the compact morula stage (P < 0.001). To determine if this was a receptor mediated effect, we attempted to rescue development of SB-75 treated embryos with a histrelin challenge. Our treatments consisted of control (n = 30), 10 μM histrelin (n = 27), 10 μM SB-75 (n = 29), and 10 μM SB-75 in combination with either 1 μM (n = 27) or 10 μM (n = 25) histrelin. Both levels of histrelin partially rescued the inhibition of blastocyst formation by SB-75 (P < 0.01). Next, we were interested in examining the signaling cascade activated following binding of GnRH to its receptor in pre-implantation embryos. Toward this end, we treated embryos with inhibitors of either PKC or PKA. First, embryos were cultured in the presence of 0 (n = 33), 0.1 (n = 35), 1 (n = 35), or 10 (n = 35) μM GF109203X (GFX), a PKC inhibitor. Similar to the results obtained with SB-75, treatment with 10 μM GFX significantly reduced development to the compact morula stage and completely blocked blastocyst formation. Second, we treated embryos (n = 15 to 17/treatment) with different concentrations (0, 0.01, 0.1, 0.5, or 1 mM) of the PKA inhibitor, SQ22536. In contrast to treatment with GFX, rates of blastocyst formation were decreased only by 35% (P < 0.05) at the highest concentration of SQ22536. The percentage of embryos developing to the hatched blastocyst stage was decreased in a dose-dependent manner following SQ22536 treatment (P < 0.05); however, this effect was not consistent with SB-75 inhibition of blastocyst formation. We suggest that GnRH has an important autocrine effect on early embryonic development, potentially signaling via PKC. Funding for M Montagner was provided by CAPES, Brazil.

scholarly journals 131 MODIFICATION OF AMINO ACID CONCENTRATIONS IN MEDIUM BY PIG EMBRYOS FROM THE ZYGOTE TO THE BLASTOCYST STAGE

2005 ◽  
Vol 17 (2) ◽  
pp. 216
Author(s):  
P. Booth ◽  
T. Watson ◽  
H. Leese
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryonic Development ◽  
Amino Acid Profile ◽  
Blastocyst Stage ◽  
Morula Stage ◽  
Amino Acid Profiles ◽  
The Difference ◽  
Amino Acid Concentrations

Pre-implantation embryos can produce and consume amino acids in a manner dependent upon stage of embryonic development (Partridge and Leese 1996 Reprod. Fert. Dev. 8, 945) that may also be predictive of subsequent viability (Houghton et al. 2002 Hum. Reprod. 17, 999). To examine these relationships in the pig, the appearance or depletion of 18 amino acids from a presumptive near-physiological mixture was determined by HPLC in porcine in vitro-produced embryos from the zygote to the blastocyst stage. Cumulus oocyte complexes derived from slaughterhouse prepubertal pig ovaries were matured for 40 h in modified TCM-199 before being fertilized (Day 0) with frozen thawed semen in tris-based medium. After 6 h, presumptive zygotes were denuded and cultured in groups of 20 in NCSU medium modified to contain a physiological mixture of 18 amino acids including 0.1 mM glutamine (NCSUaa). Groups of 2–10 embryos (dependent on stage) were removed on Day 0 (1 cell), Day 1 (2- and 4-cell), Day 4 (compact morula), and Day 6 (blastocyst) and placed in 4 μL NCSUaa for 24 h. After incubation, the embryos were removed and the medium analyzed by HPLC. Each stage was replicated 3–9 times. Since amino acid profiles of 2- and 4-cell embryos were not different, data were combined. Overall, arginine (1.19 ± 0.33), glutamine (0.78 ± 0.34) and threonine (0.05 ± 0.04) were significantly (P < 0.01) depleted from the medium whereas alanine (0.21 ± 0.1), glycine (0.20 ± 0.06), asparagine (0.13 ± 0.5), lysine (0.1 ± 0.03), isoleucine (0.08 ± 0.01), valine (0.05 ± 0.01), leucine (0.04 ± 0.02), phenylalanine (0.03 ± 0.01), and histidine (0.02 ± 0.04) significantly (P < 0.05) accumulated (mean of the 4 sampling timepoints; all values pmol/embryo/h ± SEM). The difference between amino acid accumulation and depletion (balance) was approximately equivalent between Day 0 and the morula stage although turnover (sum of depletion and accumulation) steadily decreased during this period from 3.1 on Day 0 to 1.35 pmol/embryo/h at the morula stage. However, at the blastocyst stage, turnover and balance increased to 6.32 and 2.42 pmol/embryo/h, respectively, i.e. net appearance occurred. Notable changes in amino acid profile during development included decreases in accumulation of asparagine, glutamate, and glycine in the medium and the depletion of glutamine over Days 0, 1, and 4, followed by reversal of these trends by Day 6. These data suggest that pig embryos can alter the accumulation and depletion rates of amino acids in a manner that is dependent on the specific amino acid and the stage of embryonic development. This work was supported by BBSRC.

196 A NEW APPROACH TO PREDICT MOUSE EMBRYONIC DEVELOPMENTAL COMPETENCE USING A TIME-LAPSE SYSTEM AND THE 'SHORTEST HALF' analysis procedure

2007 ◽  
Vol 19 (1) ◽  
pp. 214 ◽  
Author(s):  
S. Yavin ◽  
A. Aroyo ◽  
Z. Roth ◽  
A. Arav
Keyword(s):  
Embryonic Development ◽  
Time Lapse ◽  
Time Frame ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Embryo Morphology ◽  
Cell Stage ◽  
Developmental Potential ◽  
Additional Tool ◽  
Embryonic Cleavage

Embryonic development is a dynamic process in which embryo morphology may change immensely within several hours. Therefore, identifying and selecting embryos with the highest probability of developing and achieving a pregnancy is a major challenge. The timing of embryonic cleavage may serve as an additional indicator for the identification of quality embryos. The aim of this study was to characterize the cleavage timing of mouse embryos and to identify the stage that is most indicative of blastocyst formation. Mated mice (CB6F1) were sacrificed 20 h after hCG administration; putative zygotes were recovered and cultured (50 embryos in each 20-µL drop of M16) in a time-lapse system (EmbryoGuard; IMT, Ltd., Ness-Ziona, Israel) inside the incubator. The time-lapse system was programmed to take photos at half-hour intervals such that culture dishes were not removed from the incubator. The ‘shortest half’ statistical procedure of JMPIN (SAS Institute, Inc., Cary, NC, USA) was utilized to evaluate the period during which at least 50% of the embryonic population cleaves within the shortest time frame. Captured images made it possible to search along the time axis for the densest 50% of cleavage observations. Developing embryos were categorized into 3 groups according to the time of cleavage after hCG administration: before, during, and after the ‘shortest half’ for each developmental stage. Two hundred thirty putative zygotes cleaved and created 2-cell-stage embryos, of which 55 arrested at various stages and 175 progressed to the blastocyst stage. During embryonic development, cleavage timing appeared to become less uniform and the ‘shortest half’ became longer for each successive cell division: Whereas the shortest period in which 50% of the 2-cell-stage embryos cleaved was a 2-h interval, cleavage into the 4-cell, 8-cell, and blastocyst stages took 2.5, 3.5, and 5 h, respectively. The ‘short half’ for the first cleavage appears to be a predictive time frame for subsequent embryonic development, because cleavage was closely synchronized with 80% of the embryos developing to the blastocyst stage. Note that only a small number of embryos were actually cleaving early, while the ‘shortest half’ consisted of 50% of the embryonic population. Moreover, late-cleaving embryos in the 2-cell stage expressed inferior developmental potential relative to those that cleaved within the ‘shortest half’ (see Table 1). In summary, 2-cell-stage embryos that cleaved within the ‘shortest half’ seemed to be better synchronized and consequently more competent than the rest of the embryonic population. Embryonic cleavage timing using the ‘shortest half’ parameter can be considered a biological indicator of embryo potential. It may be useful as an additional tool for selecting embryos for transfer and cryopreservation. Table 1. Cleavage timing distribution into the 2-cell stage according to the shortest half

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).

195 SUPPRESSION OF SETDB1 DURING BOVINE EMBRYONIC DEVELOPMENT RESULTS IN PRE-IMPLANTATION MORTALITY AND DECREASED TRANSCRIPTION OF TRANSPOSABLE ELEMENTS

2015 ◽  
Vol 27 (1) ◽  
pp. 188
Author(s):  
M. D. Snyder ◽  
J. H. Pryor ◽  
K. J. Veazey ◽  
M. D. Peoples ◽  
G. L. Williamson ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Transposable Elements ◽  
Fluorescent Microscopy ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Small Interfering Rnas ◽  
Treatment Groups ◽  
Commercial Supplier ◽  
Morula Stage ◽  
Student’S T

Organization of chromatin structure by the combinatorial patterns of DNA methylation and post-translational histone modification is essential for the establishment and maintenance of proper transcriptional programs that result in the coordination of embryonic development. We previously observed that suppression of transcripts encoding SET domain, bifurcated 1 (SETDB1) using small interfering RNAs (siRNA) is embryonic lethal, with SETDB1-suppressed embryos (n = 361) arresting immediately before the blastocyst stage (blastocyst rate: Control 44.9 ± 4.9% and NULL injected 25.7 ± 6.0%). Studies in rodents indicate SETDB1 is a crucial regulator of transposable elements and that the precise epigenetic regulation of these elements is a key aspect of transcriptional programs controlling pluripotency and placentation. To better characterise the molecular basis of the observed mortality, we analysed expression of the bovine Long Interspersed Nuclear Element 1 family (LINE1) of transposable elements via quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). Mature bovine oocytes were obtained from a commercial supplier (De Soto Biosciences, Seymour, TN, USA) and IVF performed by standard laboratory protocol. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes divided into 3 different treatment groups: non-injected control (CNTL), non-targeting siRNA injected control (siNULL), and zygotes injected with siRNAs targeting SETDB1 (siSETDB1). Each embryo was injected with ~100 pL of siRNAs (10 µM) in fluorescent dextran solution. All zygotes were verified as injected by fluorescent microscopy and then cultured in Bovine Evolve (Zenith Biotech, Guilford, CT, USA) medium supplemented with 4 mg mL of BSA (Probumin, EMD Millipore, Darmstadt, Germany). Groups of embryos (15–20) from each treatment were lysed at the 4-cell, 8-cell, and morula stages, RNA extracted, and analysed by RT-qPCR using GAPDH and YWHAZ as reference genes. A two-way ANOVA and a Student's t-test were used to analyse the results from the RT-qPCR. As expected, siSETDB1-injected morulae displayed dramatic reduction in the level of Setdb1 transcripts as compared to siNULL control (96%; P < 0.05). Preliminary analysis of LINE1 transcripts at the morula stage indicated siSETDB1-injected embryos displayed a 75% reduction compared to the siNULL. Whether alteration in LINE1 regulation contributes to the developmental arrest and embryonic mortality of siSETDB1-injected embryos is under investigation.

scholarly journals Transcriptional profile of bovine preimplantation development selected based on G6PDH activity

2021 ◽  
Vol 5 (1) ◽  
pp. 001-003
Author(s):  
Ghanem Nasser ◽  
Samy Romysa ◽  
Kassab Eman Kh ◽  
Khalil Beshoy SF ◽  
Kordy Aya Ahmed ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Genetic Material ◽  
Implantation Rate ◽  
Short Review ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Blue Staining ◽  
G6pdh Activity ◽  
Cell Stage ◽  
Developmental Potential

The oocyte is the female gamete that contributes not only half of the genetic material but also all of the cytoplasm to the zygote, supplying the transcripts, proteins, mitochondria and other components necessary for early embryonic development. The intrinsic oocyte quality is one of the main factors affecting the embryo yield, the implantation rate and the rate of healthy offspring. It is obvious that a fertilized oocyte must reach the blastocyst stage within 6–9 days in the proper culture conditions to have a significant chance of inducing a pregnancy and producing an offspring. The ability to sustain the first week of embryonic development is clearly influenced by the follicular status from which the oocyte is obtained indicating that this developmental potential is inherent within certain oocytes. Since most early embryos that do not reach the blastocyst stage are blocked at or close to the maternal to zygotic transition (MZT)-stage, which occurs at the eight-cell stage in cattle, one could speculate that incompetent oocytes fail to appropriately activate the embryonic genome. Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. Recently, molecular regulation of genes regulating biological process of Brilliant Cresyl Blue staining (BCB) selected oocytes and embryos was investigated to explain their variation in quality and developmental potentiality. This short review will highlights some of these efforts that have been done in this interesting area of research.

scholarly journals 152EFFECT OF REPLACEMENT OF PYRUVATE/LACTATE IN CULTURE MEDIUM WITH GLUCOSE ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
N.W.K. Karja ◽  
S. Medvedev ◽  
D. Fuchimoto ◽  
A. Onishi ◽  
M. Iwamoto ◽  
...  
Keyword(s):  
Energy Source ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Culture Period ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Energy Substrates ◽  
Anova Test

Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) reported that replacement of pyruvate and lactate with glucose, as energy substrates, at 48h of culture in IVC medium enhanced the quality of IVP porcine blastocysts. However, the exact time during early cleavage stages when the utilization of glucose as an energy source is optimal has not yet been determined. The purpose of this study was to examine the effects of glucose supplementation at different times of culture on the developmental competence of IVP porcine embryos. Porcine cumulus-oocytes complexes were matured in modified NCSU-37 solution and fertilized in vitro according to Kikuchi et al. All cultures were performed at 38.5°C, 5% O2, 5% CO2, and 90% N2. In experiment 1, after being fertilized (Day 0), putative zygotes (1158 in 6 trials) were cultured in NCSU-37 supplemented with 0.4% BSA, 0.17mM sodium pyruvate, and 2.73mM sodium lactate (IVC-pyr/lac). Embryos (30–50 in each group) were then transferred into NCSU-37 supplemented with 0.4% BSA and 5.55mM D-glucose (IVC-glu) at 24, 48, 72, 96, or 118h of culture. As control groups, putative zygotes (391) were cultured in IVC-pyr/lac or IVC-glu for the whole culture period. In experiment 2, after being fertilized, putative zygotes (543 in 4 trials, 30–50 in each group) were cultured in IVC-pyr/lac, and then were transferred into IVC-glu at 48h, 53h, 58h, or 63h of culture, because glycolytic activity of in vitro-derived porcine embryos was reported to increase around the 8-cell stage, and some embryos develop to that stage before 72h of culture in experiment 1. All embryos were cultured for 6 days, and then development to the blastocyst stage and number of cells per blastocyst were assessed. When IVF embryos were cultured in IVC pyr/lac for 24h or 48h and subsequently in IVC-glu until day 6 in experiment 1, the rates of blastocyst formation were significantly higher (P&lt;0.05, ANOVA test) than those of embryos cultured in IVC-pyr/lac for the whole culture period (24.4% and 23.0% v. 14.5%, respectively). However, when IVC pyr/lac was replaced with IVC-glu, there were no significant differences between the energy source replacement groups and the glucose-only group in terms of the proportions of cleavage, development to the blastocyst stage and mean cell number per blastocyst (P&gt;0.05, ANOVA test) (15.2%–24.4%, and 16.8%, respectively). Replacement of pyruvate and lactate with glucose at 58h of culture in experiment 2 significantly enhanced the rate of blastocyst formation (P&lt;0.05, ANOVA test) but not the mean cell number compared with zygotes in which the replacement was done at 48, 53, and 63h of culture (31.3% v. 20.6%, 20.8%, and 21.1%, respectively) (P&lt;0.05, ANOVA test). In conclusion, replacement of pyruvate and lactate with glucose as energy substrates was optimal at 58h of culture for the in vitro development of pig embryos to the blastocyst stage.

215 INVESTIGATION OF A PREFERENTIALLY UPREGULATED GENE CLUSTER IN DAY 7 BOVINE EMBRYOS DERIVED FROM RNA SEQUENCING DATA

2013 ◽  
Vol 25 (1) ◽  
pp. 256 ◽  
Author(s):  
A. Al Naib ◽  
S. Mamo ◽  
P. Lonergan
Keyword(s):  
Rna Sequencing ◽  
Mature Oocyte ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Sequencing Data ◽  
Cell Stage ◽  
Uterine Endometrium ◽  
Embryonic Origin

Successful establishment and maintenance of pregnancy requires optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. A recent RNA sequencing study from our group (Mamo et al. 2011 Biol. Reprod. 85, 1143–1151) described the global transcriptome profile of the bovine conceptus at 5 key stages of its pre- and peri-implantation growth (Days 7, 10, 13, 16, and 19) using RNA sequencing techniques. One cluster of genes (n = 1680 transcripts) was preferentially upregulated at Day 7 and subsequently downregulated, suggesting that these genes might be markers of blastocyst formation. The objective of this study was to characterise the pattern of expression of these genes before Day 7 (i.e. from the zygote to blastocyst stage). The list of genes was submitted to DAVID (Database for Annotation, Visualisation, and Integrated Discovery) to take advantage of available ontology information contained therein. The expression of 9 genes belonging to ontologies specifically related to embryo developmental (GINS1, TAF8, ESRRB, NCAPG2, SP1, XAB2, CDC2L1, MSX1, and AQP3) plus Na/K ATPase, a gene previously known to be involved in blastocoe formation, was studied by quantitative real-time PCR (QPCR) in 6 replicate pools of 5 embryos produced by maturation, fertilization, and embryo culture in vitro. Stages studies included immature and mature oocyte, zygote, 2- cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, and hatched blastocyst. In addition, in vivo derived Day 13 and Day 16 embryos were included as controls to confirm down-regulation after Day 7. Data were analysed using the GLM procedure of SAS. The QPCR expression data supported the RNA Seq data in that expression of all transcripts was downregulated after the blastocyst stage. Expression before the blastocyst stage was characterised by 1 of 3 broad patterns: (1) the expression was of maternal origin where the expression was very high up to 8-cell stage and decreased subsequently (MSX1), (2) the expression was of embryonic origin being low up to the 8-cell stage and increasing thereafter (TAF8, ESRRB, AQP3, and Na/K ATPase), or (3) static or decreased expression from oocyte to the maternal-zygotic transition followed by increased expression from the 16-cell stage (GINS1, NCAPG2, SP1, XAB2, and CDC2L1). In conclusion, the genes identified in this cluster, despite having different patterns of expression before the blastocyst stage, may represent markers of blastocyst formation in cattle given their downregulation subsequently. Supported by Science Foundation Ireland (07/SRC/B1156).

Ultrastructural observations of lethal yellow (Ay/Ay) mouse embryos

Development ◽  
1976 ◽  
Vol 35 (1) ◽  
pp. 73-80
Author(s):  
Patricia G. Calarco ◽  
Roger A. Pedersen
Keyword(s):  
Developmental Stages ◽  
Inner Cell Mass ◽  
Structural Characteristics ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Cell Organelles ◽  
Cell Stage ◽  
Wide Range ◽  
Lethal Yellow ◽  
And Control

Ay/Ay embryos were identified by the presence of large excluded blastomeres (Pedersen, 1974) and examined cytologically and ultrastructurally. Cell organelles, inclusions and junctions in the excluded blastomeres were compared with those of non-excluded cells of Ay/Ay embryos and control embryos. Excluded blastomeres always had the fine structural characteristics of earlier developmental stages and may have arrested at the 4- to 8-cell stage or slightly later. Interior cells (inner cell mass) were observed in all mutant blastocysts. Nonexcluded cells of Ay/Ay embryos were normal until degenerative changes appear in the late blastocyst stage. The mode of action of the +Ay gene was not determined, but evidence from this study and others indicates that the effects of +Ay gene action occur over a wide range of time in early cleavage and implantation.

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