202 KNOCK-DOWN AND DEVELOPMENTAL EFFICIENCY OF Arhgap15 GENE KNOCK-DOWN EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 200
Author(s):  
B.-C. Yang ◽  
H.-C. Lee ◽  
S. Hwang ◽  
I.-S. Jeon ◽  
D.-K. Lee ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Rho Gtpase ◽  
Mrna Level ◽  
Developmental Competence ◽  
Control Group ◽  
Dependent Manner ◽  
Blastocyst Development ◽  
Knock Down ◽  
Donor Cells

The technique of RNA interference (RNAi) knock-down is a powerful tool for the analysis of gene function in mammalian cells and eggs. Rho GTPase-activating protein 15 (Arhgap15) is closely related to anaemia and immunity, especially as caused by trypanosome. This study was performed to investigate the effect of Arhgap15 gene knock-down on the developmental competence of bovine embryos in vitro. Bovine fibroblast cells were treated with 50 and 100 nM concentrations of Arhgap15 RNAi, respectively. After 24 h of transfection, the control group showed no change in Arhgap15 mRNA level, whereas mRNA expression in the RNAi-treated donor cells was obviously decreased in a dose-dependent manner. These RNAi-treated cells were then transferred into enucleated bovine oocytes to analyse the consequence of RNAi-mediated Arhgap15 gene knock-down. In the control group, cleavage and blastocyst development rates were 75% (102/136) and 14.7% (20/136), whereas those in the knock-down embryos were 81.6% (120/147) and 24.5% (36/147), respectively. The occurrence of cell death by apoptosis was examined in Day 7 blastocysts. Apoptotic cells numbered 12 ± 3.2 in control embryos and 8.9 ± 4.8 in knock-down embryos. Therefore, it can be concluded that RNAi-mediated Arhgap15 gene knock-down in somatic cells did not affect the developmental competence of bovine cloned embryos. This work was supported by grant 120080401034062 from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.

Effect of exogenous DMNPE-caged ATP on in vitro-matured bovine oocytes prior to parthenogenetic activation, fertilisation and nuclear transfer

2004 ◽  
Vol 16 (8) ◽  
pp. 781 ◽  
Author(s):  
Jun Xue ◽  
Melissa A. Cooney ◽  
Vanessa J. Hall ◽  
Natasha A. Korfiatis ◽  
R. Tayfur Tecirlioglu ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Calcium Oscillations ◽  
Developmental Competence ◽  
Control Group ◽  
Dependent Manner ◽  
Blastocyst Development ◽  
Cell Numbers ◽  
Caged Atp ◽  
Bovine Oocytes

Adenosine triphosphate (ATP) plays an important role during fertilisation of the mammalian oocyte through its ability to alter the frequency and duration of calcium oscillations. It has also been shown that higher ATP levels correlate with increased developmental competence in bovine and human oocytes. During somatic cell nuclear transfer (NT), the incoming nucleus is remodelled extensively, undoubtedly using a variety of ATP-dependent enzymes. The aim of the present study was to determine whether additional exogenous ATP influences activation of parthenogenetic (PA), in vitro-fertilised (IVF) or cloned (NT) in vitro-matured bovine oocytes. Blastocyst development and cell numbers in PA embryos were found to increase in a dose-dependent manner following the photorelease of 0, 50, 100, 500 and 1000 μm DMNPE-caged ATP (adenosine 5′-triphosphate, P3-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester, disodium salt). No cleavage was found following release of 2 and 5 mm DMNPE-caged ATP or with DMNPE-caged ATP (not photoreleased). There were also no differences in blastocyst rates or cell numbers between the control group and groups treated with caged, but not photoreleased, ATP. The addition of exogenous ATP before IVF or to NT couplets did not result in a significant increase in blastocyst development or cell number. Embryo transfer is necessary to determine whether exogenous ATP can positively affect reprogramming, resulting in higher cloned pregnancy rates or live-term births.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 118
Author(s):  
B. Gajda ◽  
Z. Smorag ◽  
M. Bryla
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Phenazine Ethosulfate ◽  
And Control

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 272
Author(s):  
K. Kananen-Anttila ◽  
M. Eronen ◽  
J. Matilainen ◽  
M. Kallio ◽  
J. Peippo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Embryo Cleavage ◽  
Bovine Oocytes ◽  
Sperm Aster

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P < 0.05; α-amanitin: 10 ± 0.6 μ­m, P < 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development

58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
I. M. Saadeldin ◽  
B. H. Kim ◽  
B. Roibas da Torre ◽  
O. J. Koo ◽  
G. Jang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Mass ◽  
Donor Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Embryonic Cells ◽  
Blastocyst Development

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.

197 EFFECTS OF PRE-IN VITRO MATURATION WITH CAFFEINE ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

2016 ◽  
Vol 28 (2) ◽  
pp. 229
Author(s):  
S. M. B. Ulloa ◽  
J. Heinzmann ◽  
D. Herrmann ◽  
U. Baulain ◽  
K.-G. Hadeler ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Developmental Competence ◽  
Differential Staining ◽  
Control Group ◽  
Dependent Manner ◽  
Meiotic Arrest ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Camp Levels

High cyclic adenosine monophosphate (cAMP) concentrations are critical for maintaining oocyte meiotic arrest in vivo. For in vitro maturation (IVM), the oocyte is released mechanically from the follicle, which induces a significant drop in intra-oocyte cAMP levels, triggering non-physiological meiotic resumption. It has been proposed that modulation of cAMP before IVM can increase bovine blastocyst rates in vitro. Caffeine is a nonspecific competitive phosphodiesterases (PDE) inhibitor and can inhibit meiotic resumption of oocytes due to maintenance of cAMP levels. It has been reported that gamete treatment with caffeine can increase developmental potential. The current study evaluated the effects of pre-in vitro maturation culture with different concentrations of caffeine on meiotic progress, developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 6648 cumulus-oocyte complexes were obtained by slicing. Caffeine was used in 5 different concentrations (1, 5, 10, 20, and 30 mM) during slicing, searching, and 2 h pre-IVM culture. A control group, with 2 h pre-IVM without caffeine (0 mM) and a standard control were also included. Oocytes were washed either after standard or pre-IVM treatments and cultured for 24 h in vitro without caffeine. After IVM, oocytes were fertilised in vitro for 19 h, and zygotes were cultured in vitro for 8 days until the blastocyst stage. Subsets of oocytes were fixed in 2% glutaraldehyde at 9, 20, and 24 h after IVM. Hoechst staining was performed to evaluate nuclear status of matured oocytes. Cleavage and blastocyst formation rates were evaluated at Days 3 and 8 after IVF. Expanded blastocysts from all treatments were submitted to differential staining. One-way ANOVA from R software was applied to evaluate differences in cleavage and blastocysts rates and blastocyst cell numbers. Fisher’s exact test complemented by Bonferroni correction was used to determine meiotic progress. Caffeine maintained oocytes in meiotic arrest after 9 h of IVM in a concentration-dependent manner (germinal vesicle: 79.0%, 92.2%, 66.7%, 55.1%, 56.9%, 43.9%, 30.2%, respectively, for 30, 20, 10, 5, 1, 0 mM and standard; P < 0.016). Cleavage rates were similar in all treatments; however 30 mM caffeine decreased blastocyst rates (Table 1; P < 0.05). The number of cells did not differ significantly among in vitro treatments (Table 1; P > 0.05). Developmental competence was not affected by 2 h pre-IVM culture. Caffeine supplementation before IVM delayed resumption of meiosis and affected embryo development. Table 1.In vitro developmental competence of oocytes treated with different caffeine concentrations before IVM

scholarly journals 237 INCREASE OF BLASTOCYST DEVELOPMENTAL RATE IN VITRO BY SELECTION OF DEVELOPMENTALLY COMPETENT COW OOCYTES BEFORE IVM USING A STAINING TEST

2005 ◽  
Vol 17 (2) ◽  
pp. 269
Author(s):  
H. Alm ◽  
H. Torner ◽  
B. Loehrke ◽  
T. Viergutz ◽  
I. Ghoneim ◽  
...  
Keyword(s):  
Developmental Rate ◽  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
G6pdh Activity ◽  
Blastocyst Development ◽  
Brilliant Cresyl Blue ◽  
Immature Oocytes ◽  
Cresyl Blue

A large proportion of bovine oocytes fail to develop to blastocyst stage following maturation, fertilization, and culture in vitro. While suboptimal culture conditions undoubtedly contribute to this poor development, it is recognized that immature oocytes, especially from cows with reduced reproductive performance or which are slaughtered on the end of their use, are heterogeneous in quality and developmental competence (Gordon 2003). The aim of the present study was to increase the efficiency of blastocyst production from cows after IVM/IVF by oocyte selection before maturation. Immature oocytes are known to synthesize a variety of proteins (Wassarman PM 1988, Annu. Rev. Biochem. 57, 415–442), among them, glucose-6-phosphate dehydrogenase (G6PDH). This enzyme is active in the growing oocyte, but has decreased activity in oocytes that have finished their growth phase. Brilliant cresyl blue (BCB) has been used to measure G6PDH activity. The BCB test is based on the capability of the G6PDH to convert the BCB stain from blue to colorless (Erisson et al. 1993 Theriogenology 39, 214). The ovaries were obtained from a slaughterhouse and transported to the laboratory; cumulus-oocyte complexes (COCs) were recovered by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control – placed immediately into culture; (2) holding control – COCs kept in PBS containing 0.4% BSA for 90 min at 38.5°C before placement into culture; and (3) treatment – incubation with brilliant cresyl blue for 90 min at 38.5°C before culture. Treated oocytes were then divided into BCB− (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction in control, BCB−, and BCB+ groups; activity was significantly increased in BCB− COCs in comparison to the control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to Day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes (77.1 and 72.5%, respectively) than for BCB− oocytes (58.1%). The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than either control group (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB− oocytes (3.9%). The number of nuclei in the blastocysts was comparable in BCB+ and both control groups (105.5 ± 5.8 and 117.5 ± 8.5, 101.8 ± 6.2, respectively). Blastocysts in the BCB− group had a significantly lower cell number (61.0 ± 2.6) than did controls. The results show that the staining of COCs from cows before IVM may be useful in increasing the efficiency of blastocyst production during standard IVF procedures. In addition, classification of G6PDH activity on the basis of BCB staining may be used to effectively select cow oocytes with further developmental competence. To our knowledge, this is the first study to evaluate the association between G6PDH activity in oocytes and further blastocyst development in cows.

scholarly journals Dielectrophoretic behavior of in vitro-derived bovine metaphase II oocytes and zygotes and its relation to in vitro embryonic developmental competence and mRNA expression pattern

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 931-946 ◽  
Author(s):  
Salilew-Wondim Dessie ◽  
Franca Rings ◽  
Michael Hölker ◽  
Markus Gilles ◽  
Danyel Jennen ◽  
...  
Keyword(s):  
Polar Body ◽  
Mrna Level ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Non Invasive ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Selection Of ◽  
Metaphase Ii Oocytes

Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase II oocytes with (PB+) and without (PB−) first polar body and zygotes were subjected to DEP at 4 MHz and 450 μm electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of post-activation, the blastocyst rate of very slow dielectrophoretic PB+and PB−oocytes was significantly (P< 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P< 0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB+oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB+oocytes and zygotes respectively. In conclusion, dielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.

Polydatin improves the developmental competence of bovine embryos in vitro via induction of sirtuin 1 (Sirt1)

2017 ◽  
Vol 29 (10) ◽  
pp. 2011 ◽  
Author(s):  
Imran Khan ◽  
Sung Woo Kim ◽  
Kyung-Lim Lee ◽  
Seok-Hwan Song ◽  
Ayman Mesalam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Group Treatment ◽  
Sirtuin 1 ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Protein Levels ◽  
Development In Vitro

The aim of the present study was to investigate the beneficial effect of polydatin (PD), the glycoside form of resveratrol, on embryo development in vitro. Oocytes were aspirated from ovaries of Korean Hanwoo cows and cultured until Day 8 in a humidified atmosphere of 5% CO2 in air at 38.5°C. Protein and gene expression levels were determined through confocal microscopy and reverse transcription–polymerase chain reaction respectively, whereas the number of total and apoptotic cells in Day 8 blastocysts was determined using Hoechst 33342 staining and terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling. Of the different concentrations of PD (0.5, 1.0 and 2.0 µM) added to the IVM medium, only 1.0 µM PD significantly improved blastocyst development. Immunofluorescence analysis confirmed that protein levels of sirtuin 1 (Sirt1) increased significantly (P < 0.05) after PD treatment, whereas levels of reactive oxygen species (ROS) were significantly (P < 0.05) decreased, as evidenced by reductions in 8-oxoguanine immunoreactivity. Similarly, protein levels of nuclear factor (NF)-κB and cyclo-oxygenase (COX)-2  were significantly (P < 0.05) lower in the PD-treated group than in the control group. Treatment with 1.0 µM PD reduced gene expression of BCL2-associated X protein, inducible nitric oxide synthase, COX2 and Nfkb, but increased the expression of Sirt1, supporting the immunofluorescence data. PD possesses antioxidant activity and is useful for embryo development in vitro. We conclude that supplementation of IVM medium with PD improves embryo developmental competence via Sirt1.

scholarly journals Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes

Reproduction ◽  
2003 ◽  
pp. 369-376 ◽  
Author(s):  
S Ikeda ◽  
H Imai ◽  
M Yamada
Keyword(s):  
Dna Fragmentation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Treated Group ◽  
Developmental Competence ◽  
Pcr Analysis ◽  
Control Group ◽  
Dependent Manner

The aim of this study was to investigate whether apoptosis occurs in cumulus cells during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes (CEOs). The bovine CEOs obtained from ovaries from an abattoir were cultured for 24 h in IVM medium in the presence or absence of 10% (v/v) fetal bovine serum. The developmental competence of enclosed oocytes, as assessed by the development of the blastocyst after IVF, was significantly higher in the serum-treated group than in the control group. The morphological features of apoptosis that were analysed by orcein staining were hardly detectable in the cumulus cells at the start (0 h) of IVM, but were evident at the end (24 h) of IVM both in the control and serum-treated groups. Genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect apoptotic internucleosomal DNA fragmentation. DNA fragmentation was hardly detectable at the start of IVM, but increased in a time-dependent manner as the IVM culture proceeded. DNA fragmentation was not observed in the oocytes, indicating that fragmentation occurs in cumulus cells. The degree of fragmentation was lower in the serum-treated group compared with the control group. The LM-PCR analysis of DNA extracted from CEOs at 24 h of IVM, in which the DNA had been pretreated with Klenow enzyme or T4 DNA polymerase, revealed that the characteristic forms of the DNA ends generated during cumulus cell apoptosis were mainly 3'-overhangs and blunt ends. In conclusion, the results of the present study demonstrate that cumulus cells in bovine CEOs spontaneously undergo apoptosis during IVM. The degree of apoptosis may be correlated with the developmental competence of the enclosed oocytes.

81 IMPROVEMENT OF DEVELOPMENTAL COMPETENCE OF BOVINE IN VITRO-PRODUCED EMBRYOS BY ADDING 2-METHOXYSTYPANDRONE IN MATURATION MEDIA

2017 ◽  
Vol 29 (1) ◽  
pp. 148
Author(s):  
A. Mesalam ◽  
I. Khan ◽  
K.-L. Lee ◽  
S.-H. Song ◽  
M.-D. Joo ◽  
...  
Keyword(s):  
Embryo Quality ◽  
Statistical Significance ◽  
Mrna Level ◽  
Cell Number ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Blastocyst Development ◽  
Sodium Pyruvate ◽  
Protein Levels

The 2-methoxystypandrone (2-MS) is a naphthoquinone isolated from Polygonum cuspidatum. The objective of this study was to investigate the effects of 2-MS on oocyte maturation, blastocyst development, and embryo quality in terms of cell number and gene expression in vitro. A total of 2364 oocytes were cultured in TCM-199 supplemented with 10% fetal bovine serum, 1 μg mL−1 oestradiol-17β, 10 μg mL−1 FSH, 10 ng mL−1 epidermal growth factor, 0.6 mM cysteine, and 0.2 mM sodium pyruvate and supplemented with different concentrations of 2-MS as following: 1.5 μM (n = 458), 1.0 (n = 493), 0.5 (n = 468), 0.1 (n = 470), and 0 μM (control, n = 475) followed by IVF and then culture in CR1-aa medium supplemented with 44 μg mL−1 sodium pyruvate, 14.6 μg mL−1 glutamine, 10 μL mL−1 penicillin-streptomycin, 3 mg mL−1 BSA, and 310 μg mL−1 glutathione for the first 3 days, and then the BSA was replaced with 10% FBS until Day 8. The differences in embryo development between experimental groups were analysed by one-way ANOVA. The Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P < 0.05. The results showed that the addition of 2-MS at 1.0 mM significantly improved (P < 0.05) the percentage of MII oocytes, which were identified by aceto-orcein staining, compared with that in the control (76.5 v. 65.4%, respectively), and remarkably (P < 0.05), improved blastocyst development rates (45.29%) compared with control (32.21%). Additionally, TUNEL assay demonstrated that treatment with 1.0 μM of 2-MS significantly improved the embryo quality by increasing total number of cells and reducing DNA damage. Immunofluorescent analysis showed that the protein levels of nuclear factor-kappa B (NFkB), inhibitor of kappa B kinase β (IkKβ), 8-oxoguanine, and cyclooxygenase-2 (COX2) declined significantly (P < 0.05) after 2-MS treatment compared with the control. These results were confirmed by qRT-PCR, which showed a significant decrease in the mRNA levels of NFkB, IkKβ, COX2, inducible nitric oxide synthase (iNOS), BCL2-associated X protein (BAX), caspase-3, and Janus kinase2 (JAK2) after 2-MS treatment; however, the mRNA level of the anti-apoptotic gene B-cell lymphoma2 (BCL2) was significantly higher than that in the control. In conclusion, the addition of 2-MS at the indicated concentration dramatically improves the developmental competence of bovine in vitro-produced embryos. This work was supported by a grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017–5), BK21plus, and KGSP.

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