239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 209
Author(s):  
T. Fanti ◽  
N. M. Ortega ◽  
R. Garaguso ◽  
M. J. Franco ◽  
C. Herrera ◽  
...  
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Basal Medium ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

168 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS DURING OOCYTE IN VITRO MATURATION ON BOVINE EMBRYOS (Gyr×HOLSTEIN) QUALITY

2016 ◽  
Vol 28 (2) ◽  
pp. 214
Author(s):  
G. R. Leal ◽  
C. A. S. Monteiro ◽  
H. F. R. A. Saraiva ◽  
A. J. R. Camargo ◽  
P. M. S. Rosa ◽  
...  
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Bovine Embryos ◽  
Tropical Conditions ◽  
Significant Difference ◽  
Number Of Cells

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr × Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Quality determines the oocyte proportion that will develop to blastocyst stage, and although the lipid content is important in oocyte development, a high concentration in embryos is associated with cryotolerance reduction, making this a relevant issue for IVP systems. The in vitro maturation system (IVM) simulated physiological oocyte maturation (SPOM) mimics the physiological maturation events by using cyclic adenosine monophosphate (cAMP) modulators, which promote the increase of oocyte competence. Among the modulators, Forskolin has lipolytic properties. The aim of this study was to evaluate the effect of the SPOM system (Albuz 2010 Hum. Reprod. 25, 12) on bovine embryos (Gyr × Holstein) regarding their total number of cells (TNC) and lipid content. Oocytes were obtained by ovum pick-up from Gyr cows in 5 replications. After selection, they were randomly divided into 2 groups: SPOM (S) and control (C). The IVM lasted 24 h for group C (TCM 199 medium without FBS) in culture oven at 38.5°C, 5% CO2 in atmospheric air and high humidity. In the SPOM system, oocytes were in pre-IVM [TCM 199 medium + 100 µM Forskolin + 500 µM 3-isobutyl-1-methylxanthine (IBMX)] for 2 h and followed for extended IVM (TCM 199 medium + 20 µM cilostamide) for 28 h under the same conditions as control group. After IVM, oocytes were fertilised with semen from a single Holstein bull that was prepared by Percoll gradient method in Fert-TALP medium (Bioklone® Animal Reproduction, São Paulo, Brazil) for 22 h and transfered to culture droplets, where they remained for 7 days (n = 10–13 per group). The lipid content analysis was performed by staining with Oil red and the stained area fraction of each embryo was measured using software ImageJ (NIH, Bethesda, MD, USA). The TNC was measured after being stained with Hoechst 33342 and results were analysed by Student's t-test in Instat GraphPad program, with a 5% significance level. There was no significant difference (P > 0.05) between embryos from both groups on TNC (group S: 88.9 ± 28.0A; group C: 101.6 ± 29.1a) and lipid content (group S: 0.93 ± 12:18A; group C: ±0.15 to 0.96) analysis. Some studies have shown there is a beneficial effect on embryo quality when using this system; however, our results demonstrated that there was no effect on total number of cells using our conditions. Some authors have also demonstrated a reduction in embryo lipid content using Forskolin during in vitro culture. Our results suggest that the time of Forskolin exposure was not enough to ensure lipolytic action on the structures produced from oocytes (Gyr) treated in pre-IVM. It was concluded that the SPOM system had no effect on TNC and lipid content of Gyr/Holstein embryos. Financial support from FAPERJ and CAPES is acknowledged.

258 IMPACT OF MILRINONE AND FORSKOLIN ON THE EFFICIENCY AND QUALITY OF BOVINE OOCYTE IN VITRO MATURATION

2013 ◽  
Vol 25 (1) ◽  
pp. 277
Author(s):  
E. Stachowiak ◽  
K. Papis ◽  
J. Karasiewicz ◽  
J. A. Modlinski
Keyword(s):  
In Vitro Maturation ◽  
Combined Treatment ◽  
Phosphodiesterase Inhibitor ◽  
Microscopic Analysis ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Experimental Conditions ◽  
Oocyte Meiosis

The efficiency of in vitro maturation (IVM) of bovine oocytes remains inferior compared with maturation in vivo. Recently, some modifications of in vitro maturation (IVM) procedures have been proposed, such as simulated physiological maturation (Gilchrist 2011 Reprod. Fertil. Dev. 23, 23–31). In our experiment, a comparison of the traditional IVM efficiency with maturation after oocyte meiosis inhibition using roscovitine or with a modified two-step maturation using forskolin (cyclic adenosine monophosphate stimulator) and milrinone (type-3 phosphodiesterase inhibitor) was performed. Control oocytes obtained from slaughterhouse-derived ovaries were subjected to the traditional 24-h maturation in TCM-199 medium supplemented with sodium pyruvate, l-glutamine, gentamicin, 10% FCS, and hormones (pregnant mare’s serum gonadotropin and hCG, PG 600, Intervet, Kenilworth, NJ, USA). The roscovitine (50 µM, 24 h) inhibitory treatment was accomplished in the same medium (without hormones) and subsequently, traditional 24-h IVM was performed. The same TCM-199 medium (with hormones) supplemented with forskolin (100 µM) and milrinone (50 µM) was used for the first step (17 h) of the two-step maturation, whereas the second step (7 h) was performed in the same TCM-199 medium devoid of forskolin and milrinone. Fertilization with frozen sperm processed using TALP media was performed in TALP supplemented with heparin, penicillamine, hypotaurine, epinephrine, and BSA. In vitro culture of presumptive zygotes was performed in CR1aa medium. Portions of oocytes from all treatments after maturation and after fertilization procedures were stained and subjected to microscopic analysis. There were no differences in terms of maturation and fertilization rates between treatments. However, roscovitine-mediated inhibition of maturation performed in our experimental conditions was efficient and reversible, but harmful for subsequent embryo development. On the other hand, two-step maturation was equally as efficient as (but not better than) traditional IVM in all aspects examined in the present study (Table 1). In conclusion, the forskolin and milrinone combined treatment during the IVM procedure gives hope for fully efficient IVM. However, to achieve this goal, more research is necessary. Table 1.Development of embryos after different oocyte maturation procedures1

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Alessandra Corallo Nicacio ◽  
Renata Simões ◽  
Fabiola Freitas de Paula-Lopes ◽  
Flavia Regina Oliveira de Barros ◽  
Maria Angelica Peres ◽  
...  
Keyword(s):  
Culture Conditions ◽  
Slow Freezing ◽  
Control Group ◽  
Hatching Rate ◽  
Rapid Freezing ◽  
Bovine Embryos ◽  
Group 10 ◽  
Controlled Freezing ◽  
Hatching Rates

SummaryThe aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7–9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen–thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

scholarly journals Cilostazol Improves Hyperglycemia-Induced Impairment of Angiogenesis by Stimulating Adiponectin/Adiponectin Receptor1/Sirtuin1

2021 ◽  
Author(s):  
Shih-Ya Tseng ◽  
Hsien-Yuan Chang ◽  
Yi-Heng Li ◽  
Ting-Hsing Chao
Keyword(s):  
Endothelial Cells ◽  
Signaling Pathway ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Antiplatelet Agent ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Sirtuin 1

Abstract Background: Cilostazol is an antiplatelet agent with vasodilating effects that functions by increasing the intracellular concentration of cyclic adenosine monophosphate. However, the effect of cilostazol on adiponectin is still unclear. Purpose: We investigated the effects of cilostazol on adiponectin/adiponectin receptors and the Sirtuin 1 (SIRT1)/AMP-activated protein kinase (AMPK) signaling pathway to prevent high glucose (HG)-induced impairment of angiogenesis in vitro and in vivo. Methods and Results: Human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were cocultured in HG conditions. Adiponectin concentrations in the supernatant were significantly increased when HASMCs were treated with cilostazol but not significantly changed when only HUVECs were treated with cilostazol. Cilostazol treatment restored the expression of the adipoR1 and SIRT1 proteins and upregulated the phosphorylation of AMPKa1 in the HUVECs treated with HG but not adipoR2. Cilostazol prevented apoptosis and stimulated proliferation, chemotactic motility and capillary-like tube formation in HG-treated HUVECs through the adipoR1/AMPK/SIRT1 signaling pathway. In cilostazol-treated mice, recovery of the blood flow ratio after hindlimb ischemia and circulating CD34+CD45dim cells were significantly attenuated by adipoR1 knockdown but not adipoR2 knockdown. The expression of SIRT1, phosphorylation of AMPKa1/acetyl-CoA carboxylase and Akt/endothelial nitric oxide synthase in ischemic muscles were significantly attenuated by gene knockdown of adipoR1. Conclusions: Cilostazol prevents HG-induced endothelial dysfunction in vascular endothelial cells and enhances angiogenesis in hyperglycemic mice by upregulating the expression of adiponectin/adipoR1 and its SIRT1/AMPK downstream signaling pathway.

Effects of Supraphysiologic Levels of Estradiol on Endometrial Decidualization, sFlt1, and HOXA10 Expression

2019 ◽  
Vol 26 (12) ◽  
pp. 1626-1632 ◽  
Author(s):  
Hanh N. Cottrell ◽  
Venkataraman Deepak ◽  
Jessica B. Spencer ◽  
Neil Sidell ◽  
Augustine Rajakumar
Keyword(s):  
Growth Factor ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Controlled Ovarian Hyperstimulation ◽  
Vascular Endothelial ◽  
Endometrial Stromal Cells ◽  
Vitro Fertilization ◽  
Endothelial Growth Factor

Objective: Supraphysiologic estradiol (E2) levels associated with controlled ovarian hyperstimulation in high in vitro fertilization (IVF) responders may alter implantation and placentation and increase the risk of preeclampsia. Our hypothesis is that elevated E2 levels in vitro significantly alter endometrial decidualization, sFlt1, and HOXA10 expression. Methods: Human endometrial stromal cells were treated with a decidualization cocktail of medroxyprogesterone, cyclic adenosine monophosphate, and 3 concentrations of E2 10 nM (standard), 100 nM (intermediate), or 1000 nM E2 (high). Effects on sFlt1, prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP-1), vascular endothelial growth factor (VEGF), and HOXA10 were studied. Results: Prolactin, IGFBP-1, and VEGF significantly increased at all 3 E2 concentrations. While IGFBP-1 and VEGF did not change with increasing E2, PRL was less with high E2 (6.0 ng/mL ± 1.4 standard error of the mean) compared to standard (21.4 ± 3.2) and intermediate (19.8 ± 3.8). sFlt1 decrease was similar at all E2 concentrations. HOXA10 was lower at standard (10%) and intermediate (30%) as expected, but did not change with high E2. Conclusions: Supraphysiologic E2 levels associated with high IVF responders that exceed in vivo levels may impair in vitro endometrial decidualization. Although PRL did increase with high E2, the levels were, however, attenuated and 3.4-fold lower than standard and intermediate E2. sFlt1 was decreased under all 3 conditions with no differences between concentrations. Reduced HOXA10 was not observed with high E2. These findings suggest that elevated E2 levels in vitro may alter endometrial decidualization and subsequently affect implantation and placentation.

A new adenylyl cyclase, putative disease-resistance RPP13-like protein 3, participates in abscisic acid-mediated resistance to heat stress in maize

2020 ◽  
Author(s):  
Hao Yang ◽  
Yulong Zhao ◽  
Ning Chen ◽  
Yanpei Liu ◽  
Shaoyu Yang ◽  
...  
Keyword(s):  
Heat Stress ◽  
Disease Resistance ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Wild Type ◽  
In Vivo Experiments ◽  
In The Wild ◽  
Shock Proteins

Abstract In plants, 3´,5´-cyclic adenosine monophosphate (cAMP) is an important second messenger with varied functions; however, only a few adenylyl cyclases (ACs) that synthesize cAMP have been identified. Moreover, the biological roles of ACs/cAMP in response to stress remain largely unclear. In this study, we used quantitative proteomics techniques to identify a maize heat-induced putative disease-resistance RPP13-like protein 3 (ZmRPP13-LK3), which has three conserved catalytic AC centres. The AC activity of ZmRPP13-LK3 was confirmed by in vitro enzyme activity analysis, in vivo RNAi experiments, and functional complementation in the E. coli cyaA mutant. ZmRPP13-LK3 is located in the mitochondria. The results of in vitro and in vivo experiments indicated that ZmRPP13-LK3 interacts with ZmABC2, a possible cAMP exporter. Under heat stress, the concentrations of ZmRPP13-LK3 and cAMP in the ABA-deficient mutant vp5 were significantly less than those in the wild-type, and treatment with ABA and an ABA inhibitor affected ZmRPP13-LK3 expression in the wild-type. Application of 8-Br-cAMP, a cAMP analogue, increased heat-induced expression of heat-shock proteins in wild-type plants and alleviated heat-activated oxidative stress. Taken together, our results indicate that ZmRPP13-LK3, a new AC, can catalyse ATP for the production of cAMP and may be involved in ABA-regulated heat resistance.

scholarly journals Ketamine Increases Proliferation of Human iPSC-Derived Neuronal Progenitor Cells via Insulin-Like Growth Factor 2 and Independent of the NMDA Receptor

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1139 ◽  
Author(s):  
Alessandra Grossert ◽  
Narges Zare Mehrjardi ◽  
Sarah J. Bailey ◽  
Mark A. Lindsay ◽  
Jürgen Hescheler ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Growth Factor ◽  
Progenitor Cells ◽  
Neural Progenitor Cells ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Nmda Receptor Antagonist ◽  
Insulin Like Growth Factor

The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine offers promising perspectives for the treatment of major depressive disorder. Although ketamine demonstrates rapid and long-lasting effects, even in treatment-resistant patients, to date, the underlying mode of action remains elusive. Thus, the aim of our study was to investigate the molecular mechanism of ketamine at clinically relevant concentrations by establishing an in vitro model based on human induced pluripotent stem cells (iPSCs)-derived neural progenitor cells (NPCs). Notably, ketamine increased the proliferation of NPCs independent of the NMDA receptor, while transcriptome analysis revealed significant upregulation of insulin-like growth factor 2 (IGF2) and p11, a member of the S100 EF-hand protein family, which are both implicated in the pathophysiology of depression, 24 h after ketamine treatment. Ketamine (1 µM) was able to increase cyclic adenosine monophosphate (cAMP) signaling in NPCs within 15 min and cell proliferation, while ketamine-induced IGF2 expression was reduced after PKA inhibition with cAMPS-Rp. Furthermore, 24 h post-administration of ketamine (15 mg/kg) in vivo confirmed phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the subgranular zone (SGZ) of the hippocampus in C57BL/6 mice. In conclusion, ketamine promotes the proliferation of NPCs presumably by involving cAMP-IGF2 signaling.

133 EFFECTS OF ACTIVINA ON mRNA EXPRESSION IN IN VITRO FERTILIZED BOVINE EMBRYOS CULTURED FROM CHEMICALLY DEFINED TWO-STEP CULTURE MEDIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 147
Author(s):  
J. E. Park ◽  
G. Jang ◽  
H. J. Oh ◽  
S. G. Hong ◽  
I. S. Yang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Early Stage ◽  
Activin A ◽  
Developmental Competence ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Defined Culture

During the preimplantation stage, embryo development occurs in a maternal environment within the oviducts and uterine horns. It has been speculated that both the embryo itself and the maternal reproductive tract provide paracrine factors that influence embryo development (Jones et al. 2006 Reproduction 132(5), 799–810). Activins are known for FSH releasers, and several previous studies have reported that activin subunits and activin receptors mRNA were expressed in oocytes, zygotes, and oviduct (Yoshioka et al. 1998 Reprod. Fertil. Dev. 10(3), 293–298; Gandolfi et al. 1995 Mol. Reprod. Dev. 40(3), 286–291). The purposes of the present study were Experiment 1) to evaluate the effects of activin A on developmental competence of bovine embryos derived from two-step defined culture medium (Lim et al. 2007 Theriogenology 67(2), 293–302) and Experiment 2) to analyze the effects of activin A on transcriptional level of the genes in IVF embryos. Cumulus–oocyte complexs were harvested from ovaries obtained from a local slaughter house, matured, and fertilized in vitro. In vitro fertilized zygotes cultured in media supplemented with activin A in the early stage at the concentrations of 0, 10, or 100 ng mL–1 or in the later stage medium at the concentrations of 0, 10, or 100 ng mL–1. Data were analyzed using the Statistical Analysis System (SAS) program. In Exp. 1, although the development competence of embryos that cultured with activin A in the early stage medium was not significantly different, development to blastocysts on day 8 in the later stage medium with 100 ng mL–1 activin A was significantly higher than the control group [22.4% (54/264) v. 34.7% (76/233); P < 0.05]. Hatching rate of blastocyst on day 8 was significantly higher in the presence of 100 ng mL–1 activin A in the later stage culture medium compared with the control group [9.3% (5/54) v. 22.4% (17/76); P < 0.05]. In Exp. 2, the relative expression of 3 genes (Na/KATPase, E-cad, Glut-1) related to blastocyst hatching and implantation was analyzed. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitative reverse transcription-polymerase chain reaction. The expression level of the Na/K ATPase, E-cad, and Glut-1 gene were higher in the presence of activin A in the culture medium compared with the control group. In conclusion, this study suggests that activin A during the later stage of in vitro bovine embryo development can enhance the developmental competence of preimplantation embryos, increase the hatching rate, and affect expression level of genes related to hatching and implantation in defined culture medium. This study was financially supported by KOSEF (grant ? M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

191 IN VITRO CULTURE DURING OOCYTE MATURATION AND FERTILIZATION INFLUENCES THE TRANSCRIPTOME PROFILES OF BOVINE BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 186
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
F. Rings ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Oocyte Maturation ◽  
Microarray Data ◽  
In Vitro Maturation ◽  
Culture Conditions ◽  
Control Group ◽  
Bovine Embryos ◽  
Vitro Fertilization

Early embryonic development, the period from oocyte maturation until blastocyst formation, is the most critical period of mammalian development. It is well known that in vitro maturation, fertilization, and culture of bovine embryos is highly affected by culture conditions. However, the stage-specific effect of culture environment is poorly understood. Therefore, we aimed to examine the effect of in vitro culture conditions during oocyte maturation and fertilization on the transcriptome profile of the resulting blastocysts. Bovine oocytes were matured in vitro and then either directly transferred to synchronized recipients, fertilized, and cultured in vivo (Vitro_M), or transferred after in vitro fertilization (Vitro_F), or at zygote stage (Vitro_Z) and blastocysts were collected at Day 7 by uterine flushing. For in vivo or in vitro fertilization, the same frozen-thawed commercial bull semen has been used. Complete in vitro (IVP) and in vivo produced blastocysts were used as controls. Gene expression patterns between each blastocyst group and in vivo produced blastocyst group were compared using EmbryoGENE's bovine microarray (EmbryoGENE, Québec, QC, Canada) over six replicates of each group (10 blastocyst/replicate). Microarray data were statistically analysed using the Linear Models for Microarray Data Analysis (LIMMA) package under the R program (The R Project for Statistical Computing, Vienna, Austria). Results showed that, the longer the embryos spent under in vitro conditions, the higher was the number of differentially expressed genes (DEG, fold-change = 2 with adjusted P-value = 0.05) compared with in vivo control group. The Vitro_M group showed the lowest number of DEG (149); in contrast IVP group represented 841, DEG, respectively compared to in vivo control group. Ontological classification of DEG showed that lipid metabolism was the most significant function influenced by in vitro maturation conditions. More than 55% of DEG in the Vitro_M group were involved in the lipid metabolism process and most of them showed down-regulation compared to in vivo control group. On the other hand, Vitro_F and Vitro_Z groups showed nearly similar numbers of DEG (584 and 532, respectively) and the majority of these genes in both groups were involved in cell-death- and cell-cycle-related functions. Pathway analysis revealed that retinoic acid receptor activation pathways were the common ones in the Vitro_M and Vitro_F groups. However, different signalling pathways were commonly dominant in the Vitro_F and Vitro_Z groups. This study provides the transcriptome elasticity of bovine embryos exposed to different environments during maturation, fertilization, and culture periods of development.

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