165 CRYOPRESERVATION OF IN VITRO PRODUCED BOVINE EMBRYOS AFTER LIPID DECREASE WITH FORSKOLIN

2016 ◽  
Vol 28 (2) ◽  
pp. 212
Author(s):  
D. Paschoal ◽  
M. Sudano ◽  
R. Maziero ◽  
M. Guastali ◽  
L. Magalhães ◽  
...  
Keyword(s):  
Lipid Content ◽  
Rate Control ◽  
Apoptotic Cell ◽  
Random Effect ◽  
Absolute Humidity ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Apoptotic Cells ◽  
Gray Intensity

Forskolin® (F-6886) is being used to induce lipolysis and increase cryotolerance, to be an activator of adenylate cyclase, and elevating the cyclic adenosine monophosphate (cAMP) levels. The objective of this experiment was to induce the chemical lipolysis of embryos to improve vitrification and the hypothesis would be that Forskolin decrease the amount of lipid droplets, improve the production of blastocysts, and increase the survival rate after vitrification and warming. Eight random effect were performed which oocytes (N = 1172) were matured in TCM 199® supplemented with 10% of fetal bovine serum (FBS), under 5% CO2 atmosphere, at a temperature of 38.5°C and absolute humidity for 24 h. Semen was selected by Percoll gradient with a final concentration of the 2 × 106 sperm mL–1. Presumptive zygotes were cultured in SOFaa and 2.5% of FBS and were kept in an incubator with 5% CO2, 5% O2 and 90% N2 at 38.5°C and absolute humidity until Day 6, when Forskolin was added and remained until Day 7; control (group without Forskolin); F 2.5 µM (group with 2.5 µM Forskolin); F 5 µM (group with 5 µM Forskolin). On Day 7 (Day 0 = IVF) the rate of blastocyst formation was observed then they were vitrified. Apoptosis was analysed using the TUNEL technique, and the lipid content analysis was performed with Sudan Black B® (S-0395). To estimate the lipid content of embryos, 1 photo at a blastocyst group was performed and submitted to the program ImageJ 1.14 (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). The embryos were limited to obtain the area (μm2), and gray intensity mean (arbitrary units), and gray intensity per area was calculated (arbitrary units/μm2). Data were analysed by ANOVA with PROC GLM of SAS (SAS Institute, Cary, NC, USA). Sources of variation in the model including treatment and replicas were regarded as fixed and random effects, respectively. Data are presented as mean and standard least-squares error. For all analyzes was adopted the significance level of 5%. There was no difference in blastocyst rate: control (37.0 ± 4.0%), F 2.5 μM (38.6 ± 4.0%), F 5 μM (40.7 ± 4.0%). There were difference in lipids content between all groups: control (136.8 ± 2.2ab); F 2.5 μM (128.5 ± 2.2b), F 5 μM (135.6 ± 2.3c; P < 0.05). The F 2.5 μM group showed the higher rate of apoptotic cells compared to other groups: control (12.1 ± 3.5%a), F 2.5 μM (16.7 ± 4.1%b), F 5 μM (11.1 ± 6.5%a; P < 0.05). After vitrification, there was no difference in re-expansion: control (71.3 ± 8.9%), F 2.5 μM (73.1 ± 8.9%); F 5 μM (66.1 ± 8.9%) and apoptosis rate: control (22.3 ± 3.1%); F 2.5 μM (37.3 ± 3.8%); F 5 μM (33.2 ± 6.5%) between the groups. The Forskolin was effective at lower concentration to diminish lipids concentrations in embryos. But when we analysed the apoptotic cell, the lower concentration of Forskolin damaged embryos, but this effect could be diminished after vitrification and warming, when the drug did not increase the apoptotic cells. However, we need to study other concentrations of Forskolin. FAPESP (2010/50410–2/2014/21289–1) is acknowledged for support.

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.

scholarly journals Pomegranate Peel Extract Is a Potential Alternative Therapeutic for Giardiasis

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 705
Author(s):  
Asmaa M. El-Kady ◽  
Iman A. M. Abdel-Rahman ◽  
Samer S. Fouad ◽  
Khaled S. Allemailem ◽  
Taghrid Istivan ◽  
...  
Keyword(s):  
Diarrheal Disease ◽  
Apoptotic Cell ◽  
In Vitro Study ◽  
Ethanolic Extract ◽  
Control Group ◽  
Pomegranate Peel ◽  
Pomegranate Peel Extract ◽  
Multiple Drugs ◽  
Peel Extract

Giardiasis is a major diarrheal disease affecting approximately 2.5 million children annually in developing countries. Several studies have reported the resistance of Giardia lamblia (G. lamblia) to multiple drugs. Therefore, identifying an effective drug for giardiasis is a necessity. This study examined the antiparasitic effect of Punica granatum (pomegranate) and evaluated its therapeutic efficacy in rats infected with G. lamblia. In vitro study showed high efficacy of pomegranate peel ethanolic extract in killing G. lamblia cysts as demonstrated by eosin vital staining. We showed that treating infected rats with pomegranate extract resulted in a marked reduction in the mean number of G. lamblia cysts and trophozoites in feces and intestine respectively. Interestingly, the number of G. lamblia trophozoites and cysts were significantly lower in the pomegranate extract-treated group compared to the metronidazole-positive control group. Moreover, pomegranate extract treatment significantly induced nitric oxide (NO) and reduced serum IL-6 and TNF-α, compared to infected untreated rats. Histological and scanning electron microscopy (SEM) examination of the jejunum and duodenum of pomegranate extract-treated animals confirmed the antiparasitic effect of the extract, and demonstrated the restoration of villi structure with reduction of villi atrophy, decreased infiltration of lymphocytes, and protection of intestinal cells from apoptotic cell death. In conclusion, our data show that the pomegranate peel extract is effective in controlling G. lamblia infections, which suggests that it could be a viable treatment option for giardiasis.

scholarly journals Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Hiroshi Yamaguchi ◽  
Toshihiko Maruyama ◽  
Yoshihiro Urade ◽  
Shigekazu Nagata
Keyword(s):  
Adenosine Receptor ◽  
Molecular Mechanisms ◽  
Apoptotic Cell ◽  
Adenosine Monophosphate ◽  
Receptor Activation ◽  
Death Process ◽  
Apoptotic Cells ◽  
Dependent Manner ◽  
A2a Adenosine Receptor ◽  
Anti Inflammatory

Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a ‘calm down’ signal.

scholarly journals Apoptotic cells induce CD103 expression and immunoregulatory function in myeloid dendritic cell precursors through integrin αv and TGF-β activation

2020 ◽  
Author(s):  
Ailiang Zhang ◽  
Helena Paidassi ◽  
Adam Lacy-Hulbert ◽  
John Savill
Keyword(s):  
Ex Vivo ◽  
Apoptotic Cell ◽  
Cell Interaction ◽  
Apoptotic Cells ◽  
Intestinal Epithelial ◽  
Mesenteric Lymph Nodes ◽  
Murine Bone Marrow ◽  
Enhanced Expression ◽  
Tgf Β1

In the mammalian gut CD103+ve myeloid DCs are known to suppress inflammation threatened by luminal bacteria, but stimuli driving DC precursor differentiation towards this beneficial phenotype are incompletely understood. We isolated CD11+ve DCs from mesenteric lymph nodes (MLNs) of healthy mice; CD103+ve DCs were 8-24 folds more likely than CD103-ve DCs to exhibit extensive of prior phagocytosis of apoptotic intestinal epithelial cells. However, CD103+ve and CD103-ve MLN DCs exhibited similar ex vivo capacity to ingest apoptotic cells, indicating that apoptotic cells might drive immature DC differentiation towards the CD103+ve phenotype. When cultured with apoptotic cells, myeloid DC precursors isolated from murine bone marrow and characterised as lineage-ve CD103-ve, displayed enhanced expression of CD103 and β8 integrin and acquired increased capacity to induce Tregs after 7d in vitro. However, DC precursors isolated from α v -tie2 mice lacking α v integrins in the myeloid line exhibited reduced binding of apoptotic cells and complete deficiency in the capacity of apoptotic cells and/or latent TGF-β1 to enhance CD103 expression in culture, whereas active TGF-β1 increased DC precursor CD103 expression irrespective of α v expression. Fluorescence microscopy revealed clustering of α v integrin chains and latent TGF-β1 at points of contact between DC precursors and apoptotic cells. We conclude that myeloid DC precursors can deploy α v integrin to orchestrate binding of apoptotic cells, activation of latent TGF-β1 and acquisition of the immunoregulatory CD103+ve β8+ve DC phenotype. This implies that a hitherto unrecognised consequence of apoptotic cell interaction with myeloid phagocytes is programming that prevents inflammation.

scholarly journals Persistence of apoptotic cells without autoimmune disease or inflammation in CD14−/− mice

2004 ◽  
Vol 167 (6) ◽  
pp. 1161-1170 ◽  
Author(s):  
Andrew Devitt ◽  
Kate G. Parker ◽  
Carol Anne Ogden ◽  
Ceri Oldreive ◽  
Michael F. Clay ◽  
...  
Keyword(s):  
Autoimmune Disease ◽  
Apoptotic Cell ◽  
Apoptotic Cells ◽  
Autoantibody Production ◽  
Apoptotic Cell Clearance ◽  
Cell Clearance ◽  
Surface Receptor ◽  
Anti Inflammatory

Interaction of macrophages with apoptotic cells involves multiple steps including recognition, tethering, phagocytosis, and anti-inflammatory macrophage responses. Defective apoptotic cell clearance is associated with pathogenesis of autoimmune disease. CD14 is a surface receptor that functions in vitro in the removal of apoptotic cells by human and murine macrophages, but its mechanism of action has not been defined. Here, we demonstrate that CD14 functions as a macrophage tethering receptor for apoptotic cells. Significantly, CD14−/− macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process. However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14−/− macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells. We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences.

scholarly journals Interaction of Apoptotic Cells with Macrophages Upregulates COX-2/PGE2and HGF Expression via a Positive Feedback Loop

2014 ◽  
Vol 2014 ◽  
pp. 1-17 ◽  
Author(s):  
Ji Yeon Byun ◽  
Young-So Youn ◽  
Ye-Ji Lee ◽  
Youn-Hee Choi ◽  
So-Yeon Woo ◽  
...  
Keyword(s):  
Positive Feedback ◽  
Tissue Repair ◽  
Feedback Loop ◽  
Apoptotic Cell ◽  
Raw 264.7 Cells ◽  
Apoptotic Cells ◽  
Positive Feedback Loop ◽  
Cox 2

Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cellsin vitroandin vivoorchestrate the interaction between COX-2/PGE2and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2production. Both NS-398 and COX-2-siRNA, as well as the PGE2receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2induction. Thein vivorelevance of the interaction between the COX-2/PGE2and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages followingin vivoexposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition.

Defective fetal liver erythropoiesis and T lymphopoiesis in mice lacking the phosphatidylserine receptor

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3362-3364 ◽  
Author(s):  
Yuya Kunisaki ◽  
Sadahiko Masuko ◽  
Mayuko Noda ◽  
Ayumi Inayoshi ◽  
Terukazu Sanui ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Apoptotic Cell ◽  
Fetal Liver ◽  
Lymphoid Cells ◽  
Cell Uptake ◽  
Developmental Defect ◽  
Apoptotic Cells ◽  
Phosphatidylserine Receptor ◽  
T Lymphopoiesis

Abstract Clearance of apoptotic cells by macrophages is considered important for prevention of inflammatory responses leading to tissue damage. The phosphatidylserine receptor (PSR), which specifically binds to phosphatidylserine (PS) exposed on the surface of apoptotic cells, mediates uptake of apoptotic cells in vitro, yet the physiologic relevance of PSR remains unknown. This issue was addressed by generating PSR-deficient (PSR-/-) mice. PSR-/- mice exhibited severe anemia and died during the perinatal period. In the PSR-/- fetal livers, erythroid differentiation was blocked at an early erythroblast stage. In addition, PSR-/- embryos exhibited thymus atrophy owing to a developmental defect of T-lymphoid cells. Clearance of apoptotic cells by macrophages was impaired in both liver and thymus of PSR-/- embryos. However, this did not induce up-regulation of inflammatory cytokines. These results indicate that during embryonic development, PSR-mediated apoptotic cell uptake is required for definitive erythropoiesis and T lymphopoiesis, independently of the prevention of inflammatory responses. (Blood. 2004;103:3362-3364)

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

165 FETAL CALF SERUM INFLUENCES CYCLIC GMP PATHWAY, WHICH IN TURN AFFECTS THE LIPID CONTENT IN IN VITRO-MATURED BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
K. R. L. Schwarz ◽  
R. C. Botigelli ◽  
F. C. Castro ◽  
M. R. Chiaratti ◽  
C. L. V. Leal
Keyword(s):  
Lipid Content ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Maturation Medium ◽  
Cgmp Pathway ◽  
Bovine Oocytes ◽  
Synthesis And Degradation

The sensitivity of IVP embryos to cryopreservation is often associated with lipid accumulation in the cytoplasm induced by the presence of fetal calf serum (FCS) during culture. Intracellular levels of cyclic (c)AMP and cGMP are involved in the regulation of lipolysis in adipocytes; high levels stimulate lipolysis whereas low levels lead to lipogenesis. Both nucleotides are present in bovine oocytes, together with the enzymes for their synthesis and degradation. The aim of this study was to analysis the effect of FCS on the cGMP pathway and the influence of cGMP on cytoplasmic lipids in bovine oocytes. In experiments 1 and 2, cumulus–oocyte complexes (COC) were cultured for 24 h in maturation medium with different proportions of FCS (2 and 10%) and a control group was matured with 0.4% BSA. After this period, transcripts for cGMP pathway were assessed by real-time PCR (GUCY1B3 and PDE5, cGMP synthesis and degradation enzymes, respectively; experiment 1) in oocytes and cumulus cells, and cGMP levels were measured in COC using commercial enzyme immunoassay kits (EIA; experiment 2). In experiments 3 and 4, COC were matured for 24 h with 0.4% BSA and different concentrations of the phosphodiesterase (PDE)5 inhibitor (0, 10–7, and 10–5 M sildenafil) to inhibit cGMP degradation and a control group was matured with 0.4% BSA. The nucleotide levels were measured in COC (experiment 3) and the oocytes were stained with Nile Red (1 μg mL–1) for evaluation of lipid content (experiment 4). Statistical analyses were performed by ANOVA followed by Tukey post hoc test using SAS software (SAS Institute Inc., Cary, NC, USA). Data for gene expression from 5 replicates and for cGMP measurements and lipid content from 3 replicates were log10-transformed into before analyses. The level of significance was 5%. The presence of FCS reduced GUCY1B3 expression in both cells and increased PDE5A in cumulus cells (P < 0.05). In experiment 2, the groups treated with 2 (0.64 fmol/COC) and 10% FCS (1.04 fmol/COC) showed decreased cGMP levels compared with control (9.46 fmol/COC; P < 0.05). In experiment 3, inhibition of PDE5A increased cGMP levels in the treated groups (36 and 56 fmol/COC for 10–7 and 10–5 M sildenafil, respectively) compared with control (9.5 fmol/COC; P < 0.05). Therefore, sildenafil showed inverse effects compared with FCS (experiment 2). In experiment 4, oocytes treated with 10–7 and 10–5 M sildenafil showed a reduced lipid content compared with controls (11.6 ± 9.4 v. 13.9 μm2 fluorescence intensity, respectively; P < 0.05). The results suggest that FCS in maturation medium affects the cGMP pathway, interfering with the transcription of genes that control its levels, which in turn results in nucleotide reduction. Inhibition of PDE5 increases cGMP levels and reduces the lipid content of oocytes, indicating that changes in this pathway caused by FCS may affect lipid metabolism of oocytes. More studies are underway to better understand this mechanism. The authors acknowledge FAPESP 2012/00170-0 for financial support.

131 APOPTOSIS AND DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS EXPOSED TO SUPRAPHYSIOLOGICAL CONCENTRATIONS OF INSULIN-LIKE GROWTH FACTOR-1 (IGF-1)

2009 ◽  
Vol 21 (1) ◽  
pp. 165
Author(s):  
M. A. Velazquez ◽  
H. Niemann
Keyword(s):  
Laser Scanning ◽  
Apoptotic Cell ◽  
Polycystic Ovary ◽  
Statistical Significance ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Control Group ◽  
Fisher Exact Test ◽  
Bovine Embryos

It has been hypothesized that high non-physiological IGF-1 levels are partially responsible for the recurrent pregnancy loss observed in women with the polycystic ovary syndrome (Eng GS et al. 2007 Diabetes 56, 2228–2234). The aim of this study was to determine the effect of supraphysiological concentrations of IGF-1 on blastocyst production and the occurrence of apoptosis in bovine embryos, which are a good model for human embryo development (Baumann CG et al. 2007 Mol. Reprod. Dev. 74, 1345–1353). COC obtained by slicing from abattoir ovaries were matured (TCM-199, Sigma) for 24 h and fertilized (Fert-TALP) for 18 h (Day 0) in vitro. Two different IGF-1 (Recombinant human IGF-1, R&D Systems GmbH, Wiesbaden, Germany) concentrations (supraphysiological = 1000 ng mL–1 and physiological = 100 ng mL–1) were added to the culture media (Synthetic oviduct fluid/BSA) and compared with a control group (no IGF-1 supplementation). On Day 8, blastocyst rates (22 replicates) were recorded and DNA degradation was detected in blastocyst nuclei using a cell death detection kit (Roche Diagnostics GmbH, Mannheim, Germany) based on the terminal deoxinucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) principle. Embryos (n = 27 [control], n = 29 [both IGF-1 groups]) from 4 replicates were examined by confocal laser scanning microscopy. Data were analyzed by ANOVA and the Fisher exact test using the SigmaStat 2.0 software package (Jandel Scientific, San Rafael, CA). Cleavage was numerically improved by both, 1000 (59.1 ± 1.8) and 100 (58.2 ± 2.8) ng IGF-1 over controls (53.5 ± 2.2), but the differences did not reach statistical significance (P = 0.22). The proportion of hatched blastocysts was enhanced by 100 (5.8 ± 1.0, P = 0.03) and 1000 (5.1 ± 0.7, P = 0.03) ng IGF-1 compared to controls (2.8 ± 0.6). Total blastocyst rate was increased by 100 ng IGF-1 (34.4 ± 1.9, P = 0.02) over controls (28.3 ± 1.7), but not by 1000 ng IGF-1 (29.1 ± 1.6 P = 0.75). The 100 ng IGF-1 group (38.5 ± 3.7) had fewer degenerated embryos (P = 0.01) compared to 1000 ng IGF-1 (49.7 ± 3.3). The proportion of embryos displaying at least one apoptotic cell was greater in the 1000 ng IGF-1 group over controls (96% v. 77% P = 0.04). The number of blastomeres with TUNEL-positive nuclei per embryo was higher in the supraphysiological group (5.5 ± 0.6, P < 0.001) compared with the control (2.3 ± 0.4) and the physiological group (2.5 ± 0.3). There were no significant differences between the control and the 100 ng IGF-1 group in this regard (P = 0.49). In conclusion, supraphysiological concentrations of IGF-1 do not increase blastocyst production but increase levels of apoptosis in bovine embryos produced in vitro. M. A. V. is in the PhD program of the University of Veterinary medicine, Hannover, Germany, and is supported by the German Academic Exchange Service (DAAD)

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