scholarly journals In Vitro Maturation with Leukemia Inhibitory Factor Prior to the Vitrification of Bovine Oocytes Improves Their Embryo Developmental Potential and Gene Expression in Oocytes and Embryos

2020 ◽  
Vol 21 (19) ◽  
pp. 7067
Author(s):  
Meritxell Vendrell-Flotats ◽  
Tania García-Martínez ◽  
Iris Martínez-Rodero ◽  
Manel Lopez-Bejar ◽  
Jonathan LaMarre ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Embryo Development ◽  
Leukemia Inhibitory Factor ◽  
In Vitro Maturation ◽  
Expression Patterns ◽  
Inhibitory Factor ◽  
Blastocyst Development ◽  
Developmental Potential

Oocyte cryopreservation has a significant impact on subsequent embryonic development. Herein, we investigated whether supplementing in vitro maturation medium with Leukemia Inhibitory Factor (LIF) prior to vitrification affects embryo development and gene expression at different embryo developmental stages. A panel of genes including maternal effect, epigenetics, apoptosis and heat stress was relatively quantified. The results show reduced cleavage rates after vitrification, regardless of the LIF treatment. Although not statistically different from control-vitrified oocytes, oocyte apoptosis and the blastocyst yield of LIF-vitrified oocytes were similar to their non-vitrified counterparts. Vitrification increased oocyte ZAR1, NPM2 and DPPA3 gene expression while its expression decreased in LIF-vitrified oocytes to similar or close levels to those of non-vitrified oocytes. With a few gene-specific exceptions, vitrification significantly increased the expression of DNMT3A, HDAC1, KAT2A, BAX and BCL2L1 in oocytes and most stages of embryo development, while comparable expression patterns for these genes were observed between LIF-vitrified and non-vitrified groups. Vitrification increased HSPA1A expression in oocytes and HSP90AA1 in 2-cell embryos. Our data suggest that vitrification triggers stage-specific changes in gene expression throughout embryonic development. However, the inclusion of LIF in the IVM medium prior to vitrification stimulates blastocyst development and several other developmental parameters and induces oocytes and embryos to demonstrate gene expression patterns similar to those derived from non-vitrified oocytes.

282 GENE EXPRESSION PATTERNS AND QUALITY OF BOVINE EMBRYOS PRODUCED UNDER DIFFERENT OXYGEN CONCENTRATIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 256
Author(s):  
W. J. Son ◽  
M. K. B. ◽  
Y. J. Jeong ◽  
S. Balasubramanian ◽  
S. Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Patterns ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Development ◽  
Glut 1

Various factors are known to influence the survival and development of in vitro-produced embryos, including co-culture with somatic cells, antioxidants, and O2 tension. Studies in several species report that embryo development and quality were enhanced at low O2 concentrations. This study compared the effects of 2 O2 concentrations on IVP embryo development, embryo quality, and gene expression to those of in vivo counterparts. Cumulus–oocyte complexes were matured in vitro in TCM-199 with hormones and 10% FCS, and inseminated in TALP medium. Presumptive zygotes were cultured in SOF medium under either 5% or 20% O2 in air. In triplicate, sets of 5 embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and Day 7 blastocyst stages were used for analyzing the expression patterns of apoptotic (Bax and Bcl2), metabolism (Glut-1 and Glut-5), stress (Sox, Hsp70, and G6PDH), compaction (Cx43), oxidation (PRDX5, NADH, and MnSOD), and implantation (VEGF and IFN-tau) genes using real-time quantitative PCR. The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Statistical analysis was performed with Bonferroni and Duncan tests by ANOVA (P < 0.05). Cleavage rates did not differ among groups. Blastocyst and hatched blastocyst development in 5% O2 was significantly (P < 0.05) higher than in 20% O2. Total cell number of in vivo blastocysts was significantly (P < 0.05) higher than that of IVP blastocysts. ICM ratio and apoptosis of in vivo blastocysts were significantly (P < 0.05) lower than for IVP blastocysts. The relative abundances (RAs) of Glut-1, Glut-5, MnSOD, NADH, PRDX5, Cx43, Bcl2, and IFN-τ were significantly (P < 0.05) higher in in vivo embryos, whereas the RAs of Sox, G6PDH, Hsp70, Bax, and VEGF were significantly (P < 0.05) lower than for IVP counterparts. In conclusion, culture at 5% O2 concentration resulted in higher rates of development to the blastocyst stage, higher total cell numbers, and decreased apoptosis. Furthermore, differences in expression of genes including Glut-1, Glut-5, Sox, G6PDH, Hsp70, Bax, Bcl2, Cx43, PRDX5, NADH, MnSOD, VEGF, and IFN-τ may prove useful in determining optimal culture conditions. This work was supported by ARPC (204119-03-SB010), Republic of Korea.

scholarly journals 296 LEUKEMIA INHIBITORY FACTOR INFLUENCES SHEEP OOCYTE PARTHENOGENETIC DEVELOPMENT DURING THE TRANSITION FROM GERMINAL VESICLE TO EARLY PRONUCLEAR STAGE

2005 ◽  
Vol 17 (2) ◽  
pp. 298
Author(s):  
G. Ptak ◽  
F. Lopes ◽  
P. Loi
Keyword(s):  
Embryo Development ◽  
Leukemia Inhibitory Factor ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Inhibitory Factor ◽  
Control Group ◽  
Blastocyst Development ◽  
Human Follicular Fluid ◽  
Oocyte Competence

Leukemia inhibitory factor (LIF) is an indispensable cytokine for female fertility. The influence of LIF on embryo development and particularly implantation has been recently confirmed; however, the effect of this cytokine on the oocyte has not been studied. The presence of LIF in human follicular fluid implies its possible role in the acquisition of oocyte competence. Furthermore, the up-regulation of LIF by steroid hormones in sheep makes entirely feasible the hypothesis that ovulatory estradiol peak plays a role in the preparation of female gamete for fertilization. With this in mind, we studied the effect of LIF during in vitro development of sheep oocytes mimicking the physiological expression of LIF induced by the ovulatory peak of estradiol in mice. GV stage oocytes matured and chemically activated in the presence of LIF and anti-LIF antibody were cultured to the blastocyst stage in our standard media. To eliminate the effect of the putative presence of LIF in heat inactivated fetal calf serum used for oocyte maturation, aliquots of LIF were treated at 56°C for 30 min and added to the maturation medium. The proportion of embryos that reached the blastocyst stage in vitro was significantly higher (P < 0.001) for oocytes matured and activated with LIF (36/93; 39%) than for the group incubated with antibody against LIF (6/68; 9%). The significant effect of anti-LIF antibody (P < 0.001) was also observed when compared with blastocysts developed from the control group of oocytes matured without LIF addition (31/106; 29%). Although the beneficial influence of LIF treatment on embryo development demonstrated with those preliminary data was not confirmed statistically, due to low number of oocytes involved, the proportion of embryos reaching the blastocyst stage in vitro was about 10% higher for those incubated with LIF than for either those cultured without the cytokine or those, matured in the presence of heat-treated LIF (15/55; 27%); however, the rate of blastocyst development appeared very similar to that of the control group. This study revealed for the first time a role of LIF in determining oocyte competence. Further investigation to determine how LIF achieves its effects on the oocyte are ongoing in our laboratory. This work was supported by FIRB RBNE01HPMX, COFIN 2002074357, COFIN 2003073943 002, and British Council 2004.

Use of a novel polydimethylsiloxane well insert to successfully mature, culture and identify single porcine oocytes and embryos

2014 ◽  
Vol 26 (3) ◽  
pp. 375 ◽  
Author(s):  
Ye Yuan ◽  
Melissa Paczkowski ◽  
Matthew B. Wheeler ◽  
Rebecca L. Krisher
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
In Vitro Maturation ◽  
Transcript Abundance ◽  
Blastocyst Development ◽  
Developmental Potential

The objective of this study was to evaluate the efficacy of a novel polydimethylsiloxane (PDMS) well-insert system for oocyte in vitro maturation (IVM) and in vitro embryo culture (IVC) in pigs. The PDMS well inserts, consisting of multiple microwells with connecting microchannels, resulted in equivalent blastocyst development compared with standard microdrop culture for IVC. These PDMS well inserts were then evaluated for IVM or IVC in a rocking versus static environment. The rocking environment during both oocyte IVM and embryo culture had detrimental effects on oocyte and embryo development compared with a static environment. Importantly, blastocyst development of oocytes and embryos cultured in the PDMS well inserts in the static environment was equivalent to that of standard microdrops. Further analysis of transcript abundance in blastocysts produced from these different environments revealed that the PDMS well-insert system may produce more viable embryos. In conclusion, this PDMS well-insert system can successfully mature oocytes and culture embryos in an individually-identifiable manner without compromising, and perhaps enhancing, developmental potential.

189 THE EFFECTS OF RESVERATROL ON PORCINE OOCYTES IN VITRO MATURATION AND SUBSEQUENT DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

2012 ◽  
Vol 24 (1) ◽  
pp. 207 ◽  
Author(s):  
S. S. Kwak ◽  
S. A. Jeong ◽  
Y. B. Jeon ◽  
S. H. Hyun
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Control Group ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Significant Difference

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, gene expression in matured oocytes and subsequent embryonic development after parthenogenetic activation (PA) and IVF. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. In experiment 1, a total of 1146 cumulus–oocyte complexes (COC) were divided into 5 groups (0, 0.1, 0.5, 2.0 and 10.0 μM resveratrol). In the nuclear maturation after 44-h IVM, the groups of 0.1, 0.5 and 2.0 μM (83.0, 84.1 and 88.3%, respectively) had no significant difference compared to the control group (84.1%). The group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (P < 0.05). In experiment 2, a total of 300 matured oocytes were examined for the effects of different resveratrol concentrations (0, 0.5, 2.0 and 10.0 μM) on porcine oocyte intracellular GSH and ROS levels. The groups of 0.5 and 2.0 μM showed a significant (P < 0.05) increase in intracellular GSH levels (1.3 and 1.3, respectively) compared with the control and 10.0 μM groups (1.0 and 1.0, respectively). The intracellular ROS level of oocytes matured with 2.0 μM resveratrol (0.4) was significantly (P < 0.05) decreased compared to other groups (control: 1.0; 0.5 μM: 0.6; and 10.0 μM: 0.7). In experiment 3, lower expression of apoptosis-related genes (Bax, Caspase-3 and Bak) was observed in matured oocytes treated with 2.0 μM resveratrol when compared with that of the control (P < 0.05). In experiment 4, a total of 728 oocytes were divided into 4 groups (control, 0.5, 2.0 and 10.0 μM) and examined subsequent to embryonic development after PA. Oocytes treated with 2.0 μM resveratrol during IVM had a significantly higher cleavage (CL) rate, blastocyst (BL) formation rate and total cell numbers (TCN) after PA compared with those of the control (2.0 μM: 96.6%, 62.1% and 49.1 vs control: 88.3%, 48.8% and 41.4, respectively) and the 10.0 μM groups (87.3%, 41.4% and 40.9, respectively). Oocytes treated with 0.5 μM resveratrol (87.2%, 50.5% and 48.6, respectively) during IVM had significantly higher TCN, but there were no differences in CL and BL formation rates. In experiment 5, a total of 935 oocytes in 3 groups (control, 2.0 and 10.0 μM resveratrol) were conducted in IVF. The BL formation rate and TCN were significantly higher in the group of 2.0 μM resveratrol (20.5% and 54.0, respectively) than the control (11.0% and 43.4, respectively) and 10.0 μM group (11.7% and 45.0, respectively), but there was no significant difference in CL rate. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH concentration, decreasing the ROS level and decreasing apoptosis-related gene expression during oocyte maturation. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008121), Rural Development Administration, Republic of Korea.

341 OXYGEN TENSION AND EGF AFFECT METABOLISM OF IN VITRO-MATURED CUMULUS-ENCLOSED MOUSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 278
Author(s):  
K. A. Preis ◽  
G. E. Seidel Jr ◽  
D. K. Gardner
Keyword(s):  
Glucose Uptake ◽  
Oxygen Tension ◽  
Embryo Development ◽  
Metabolic Activity ◽  
Lactate Production ◽  
Defined Medium ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Exact Test

In vitro maturation of immature oocytes results in limited success in both clinical and research laboratories. Although reduced oxygen concentration is beneficial to embryo development, the optimal concentration for oocyte maturation has yet to be determined. The objective of this study was to determine whether oxygen tension (20% or 5% O2) affects oocyte physiology. Additionally, the effect of epidermal growth factor (EGF) in maturation medium on oocyte metabolic activity and subsequent embryo development was determined. Cumulus–oocyte complexes (COCs; n = 231) were collected from 28-day-old unprimed F1 (C57BL/6 × CBA/ca) mice. COCs were individually matured in defined medium at 37°C in 6% CO2 in one of four groups (Table 1). For the metabolism study, COCs were further divided into two groups: individual maturation in a 2-µL drop of medium for 16 h (n = 131); or individual maturation in 5-μL for 12 h and then placed in a 0.5-μL drop of medium for 4 h (n = 100), the time of greatest metabolic activity of the COC. At 17 h of maturation, COCs were individually fertilized, and zygotes were individually cultured until 96 h, at which time blastocyst development was assessed. Metabolic profiles were analyzed by ANOVA, and blastocyst rates were analyzed by Fisher's exact test. Maturation rates and blastocyst development were not different between groups. However, at 12–16 h of maturation, metabolism of COCs was affected by both oxygen tension and EGF (Table 1). Concerning metabolism over the entire course of maturation, glucose uptake and lactate production were higher in COCs in 5% O2 + 100 ng EGF (P < 0.05) than in the remaining three groups. There was no difference between 5% O2 and 20% O2 + 100 ng EGF, but 20% O2 caused less glucose uptake and lactate production than did the other three treatment groups (P < 0.05). Results of this study are the first to show that oxygen tension alters COC metabolism: COCs matured under 5% O2 were more active metabolically than COCs matured under 20% O2. The effect of oxygen tension is to some extent moderated by the presence of EGF, as metabolic activity of COCs matured under 20% O2 + 100 ng EGF was closer to that of COCs matured under 5% O2 conditions. Although blastocyst rates were similar across the four groups, embryos derived from oocytes matured in different oxygen tensions may exhibit different developmental potential. In conclusion, results of this study have implications for the improvement of maturation conditions in both clinical and research laboratories. Table 1. Carbohydrate metabolism of individual COCs at 12–16 h of maturation

53 EFFECT OF THE TIME INTERVAL BETWEEN OVARY COLLECTION AND OOCYTE IN VITRO MATURATION ON EQUINE CLONED EMBRYO DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 184
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
R. Olivera ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Limiting Factor ◽  
Time Interval ◽  
Activation Process ◽  
Blastocyst Development ◽  
Sodium Pyruvate ◽  
Chi Square ◽  
Group I

The availability of viable equine oocytes is a limiting factor on in vitro embryo production; therefore, it is necessary to assess some of the variables that affect oocyte viability. The aim of our study was to evaluate one of those variables: the effect of time between the collection of the ovary and oocyte in vitro maturation. Ovaries of slaughtered mares were collected during the breeding season (Argentine, Southern hemisphere). They were separated in bags every half hour and treated separately after arriving at the laboratory. COCs were recovered by a combination of scraping and washing of all visible follicles with a syringe filled with DMEM supplemented with 1 mM sodium pyruvate and 15 IU mL-1 heparin. COCs were matured for 24 to 26 h in 3 groups, according to time interval: 4 to 7 (group I), 7 to 10 (II), and 10 to 12 (III) hours. The medium for maturation was TCM-199 supplemented with 10% fetal bovine serum (FBS), 1 μL mL-1 insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine, and 0.1 mg mL-1 of FSH at 39°C in a humidified atmosphere of 5% CO2 in air. The cumulus was removed by a trypsin treatment and vortexing in hyaluronidase (1 mg mL-1). Cloning and fusion procedures were performed following the zona-free technique described by Lagutina et al. (2007 Theriogenology 67, 90-98). Two experiments were carried out by using different activation protocols. In experiment 1, the activation process was 22 mM ionomycin in H-TALP for 4 min followed by 3h culture in 1.9 mM 6-DMAP in SOF, whereas in experiment 2, we used 8.7 mM ionomycin in H-TALP for 4 min followed by 4 h culture in 1 mM 6-DMAP and 10 mg mL-1 cycloheximide in SOF. Embryos were cultured in wells of well (WOW) system. Half of the medium was renewed on Day 3 with fresh SOF and on Day 5 with DMEM/F12 with 10% FBS. Cleavage was assessed 48 h after activation; the rate of blastocyst formation was recorded at Days 8 and 9. Results were compared using chi-square test (P < 0.05). In experiment 1, maturation rates were significantly different between group I (n = 135, 54.1%) and III (n = 94, 40.4%), group II did not differ from them (n = 138, 53%). Cleavage rates differed statistically between II (n = 44, 75%) and III (n = 27, 40.7%), but not with group I (n = 53, 98%). No significant differences were found in blastocyst development; however, we observed a certain tendency towards an increase in the blastocyst rate as the time interval was lower (I: 3/53, 5.7%; II: 1/44, 2.3%; III: 0/27, 0%). In experiment 2, there were no significant differences between group I and II in rates of maturation (n = 56, 59% v. n = 111, 44.5%), cleavage (n = 22, 91% v. n = 34, 82%) or blastocyst rates (1/22, 4.5% v. 7/34, 20.6%). We conclude that cloned equine embryo development, using the two activation protocols tested, is not affected when the time interval between ovary collection and oocyte IVM is within 4 to 10 h.

233. Oxygen concentration during in vitro maturation of murine oocytes affects blastocyst cell lineage

2005 ◽  
Vol 17 (9) ◽  
pp. 91
Author(s):  
K. M. Banwell ◽  
M. Lane ◽  
D. L. Russell ◽  
K. L. Kind ◽  
J. G. Thompson
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Cell Lineage ◽  
Developmental Competence ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Significant Difference ◽  
O2 Concentration

Follicular antral oxygen tension is thought to influence subsequent oocyte developmental competence. Despite this, in vitro maturation (IVM) is routinely performed in either 5 or 20% O2 and while low O2 has been shown to be beneficial to embryo development in many species, the effect of altering O2 concentration during IVM has not been adequately investigated. Here we investigated the effects of a range of O2 concentrations during IVM on meiotic maturation and subsequent embryo development after IVF. Ovaries from eCG-stimulated CBA F1 female mice (21 days) were collected and intact cumulus oocyte complexes (COCs) cultured for 17–18 h under 2, 5, 10 or 20% O2 (6% CO2 and balance of N2). Matured COCs were denuded of cumulus cells, fixed and stained (1% aceto-orcein) for visualisation of maturation status. No significant difference in maturation rates between treatment groups was observed. Following IVF (performed under 5% O2, 6% CO2 and balance of N2), no difference in fertilisation rates between treatment groups was observed in a randomly selected cohort 7 h post-fertilisation. There was also no significant difference in cleavage rates after 24 h or ability to reach blastocyst stage after 96 h, with a tendency (P = 0.079) for more blastocysts in 2% O2. However there was a significant increase in the number of trophectoderm cells present in the resulting blastocysts (P < 0.05) in the 2% O2 group (35 ± 2.1) compared to 20% O2 (25 ± 2.8). Our data suggests that O2 concentration during IVM does not influence nuclear maturation or subsequent fertilisation, cleavage and blastocyst development rates. However, maturation in 2% O2 significantly alters subsequent cell lineage within blastocysts to favour trophectoderm development. Such skewed trophectoderm cell number may influence embryo viability. Funded by NHMRC and NIH.

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.

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